Compositions and methods of improving specificity in genomic engineering using rna-guided endonucleases

ABSTRACT

Disclosed herein are optimized guide RNAs (gRNAs) that have increased target binding specificity and reduced off-target binding. Further disclosed herein are methods of designing and using the optimized gRNAs.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 15/754,861, filed Feb. 23, 2018, which is a national stage filing under 35 U.S.C. § 371 of International Patent Application No. PCT/US2016/048798, filed Aug. 25, 2016, which claims priority to U.S. Provisional Application No. 62/209,466, filed Aug. 25, 2015, the entire contents of each of which are hereby incorporated by reference.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with Government support under Federal Grant Nos. MCB1244297 and CBET1151035 awarded by the National Science Foundation and F32GM11250201, R01DA036865, and DP20D008586 awarded by the National Institutes of Health. The Government has certain rights to this invention.

SEQUENCE LISTING

The instant application includes a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Jul. 15, 2022, is named 028193-9240-US02.xml and is 465,156 bytes in size.

TECHNICAL FIELD

The present disclosure is directed to optimized guide RNAs (gRNAs) and methods of designing and using said gRNAs that have increased target binding specificity and reduced off-target binding.

BACKGROUND

RNA-guided endonucleases, notably the protein Cas9, have been hailed as a potential “perfect genomic engineering tool” because they can be directed by a single ‘guide RNA’ molecule to cut DNA with nearly any sequence. This ability has been recently exploited for a number of emerging biological and medical applications, generating tremendous excitement and promise for their future use. However, practical genomic engineering requires extremely precise control over the ability to target selectively and cut precise DNA sequences, lest off-target DNA become inadvertently damaged and mutated.

Cas9 is the endonuclease of the prokaryotic type II CRISPR (clustered, regularly interspaced, short palindromic repeats)—CRISPR-associated (Cas) response to invasive foreign DNA. During this response, Cas9 is first bound by a CRISPR RNA (crRNA):trans-activating crRNA (tracrRNA) duplex, and then directed to cleave DNA that contain 20 basepair (bp) ‘protospacer’ sites complementary to a variable 20 bp segment of the crRNA (FIG. 1A). Having bound a single-guide RNA (sgRNA), the Cas9-sgRNA complex binds to 20 bp ‘protospacer’ sequences in targeted DNA, provided that the protospacer is directly followed by a protospacer adjacent motif (PAM, here ‘TGG’). Following binding, the Cas9 endonuclease produces double-strand breaks (triangles) within the protospacer. Essentially, the only constraint on sequences that Cas9 can target is that a short protospacer adjacent motif (PAM), such as ‘NGG’ in the case of S. pyogenes Cas9, must immediately follow the protospacer sites in the foreign DNA molecule. An analysis of crystallographic and biochemical experiments suggests that specificity in protospacer binding and cleavage is imparted first through the recognition of PAM sites by Cas9 protein itself, followed by strand invasion by the bound RNA complex and direct Watson-Crick base-pairing with the protospacer (FIG. 1A).

Cas9's ability to be modularly ‘programmed’ by a single RNA hairpin to target nearly any DNA site has recently generated tremendous excitement after CRISPR-Cas9 systems were re-appropriated for a number of heterologous biotechnological applications. Notably, a single-guide RNA (sgRNA) hairpin has been designed which combine the essential components of crRNA:tracrRNA duplexes into single functional molecules. With this sgRNA, Cas9 can be introduced into a variety of organisms to produce targeted double strand breaks in vivo for remarkably facile genomic engineering. Nuclease-null Cas9 (D10A/H840A, known as ‘dCas9’) and chimeric dCas9 derivatives have also been used to alter gene expression via targeted binding at or near promoter sites in vivo as well as to introduce targeted epigenetic modifications.

Off-target binding and cleavage by Cas9 is a concern as it can adversely affect its potential uses in practice. Significant efforts have been made to improve specificity of Cas9/dCas9 activity. First, the most widespread effort is largely accomplished through intelligent selection of target sequences without similar other sequences in the genome, although a recent survey found that these methods performed poorly in their ability to predict off-target cleavage. Additionally, efforts have also been made to directly engineer the protein itself, through introduction of point mutations which were found to modulate or increase specificity in PAM or protospacer binding. Cas9 derivatives which only nick a single strand of DNA rather than perform double stranded DNA cleavage are also used in pairs (‘paired nickases’), with the assumption that the probability that off-target nicking at multiple sites that are close enough to each other to produce a double-strand break would be extremely rare. Finally, there has been some work in producing guide RNA variants themselves in an attempt to achieve greater specificity. Earlier efforts where 5′-extensions to guide RNAs were added in order to complement additional nucleotides beyond the protospacer did not show increased Cas9 cleavage specificity in vivo. Rather, they were digested back approximately to their standard length in living cells (FIG. 1A). For applications in genomic engineering, particularly for therapeutic applications, extreme specificity in the gene targeting is required, lest off-target DNA be damaged and unauthorized mutations occur. However, there have been several reports of off-target binding and cleavage by Cas9, which can adversely affect its potential uses in practice.

There remains a need for reducing off-target binding and increasing nuclease specificity using the CRISPR/Cas9 system.

SUMMARY OF THE INVENTION

The present invention is directed to a method of generating an optimized guide RNA (gRNA). The method comprises: a) identifying a target region of interest, the target region of interest comprising a protospacer sequence; b) determining a polynucleotide sequence of a full-length gRNA that targets the target region of interest, the full-length gRNA comprising a protospacer-targeting sequence or segment; c) determining at least one or more off-target sites for the full-length gRNA; d) generating a polynucleotide sequence of a first gRNA, the first gRNA comprising the polynucleotide sequence of the full-length gRNA and a RNA segment, the RNA segment comprising a polynucleotide sequence having a length of M nucleotides that is complementary to a nucleotide segment of the protospacer-targeting sequence or segment, the RNA segment is at the 5′ end of the polynucleotide sequence of the full-length gRNA, the first gRNA optionally comprising a linker between the 5′ end of the polynucleotide sequence of the full-length gRNA and the RNA segment, the linker comprising a polynucleotide sequence having a length of N nucleotides, the first gRNA capable of invading the protospacer sequence and binding to a DNA sequence that is complementary to the protospacer sequence and forming a protospacer-duplex, and the first gRNA capable of invading an off-target site and binding to a DNA sequence that is complementary to the off-target site and forming an off-target duplex; e) calculating an estimate or computationally simulating the invasion kinetics and lifetime that the first gRNA remains invaded in the protospacer and off-target site duplexes, wherein the dynamics of invasion are estimated nucleotide-by-nucleotide by determining the energetic differences between further invasion of a different gRNA and re-annealing of the first gRNA to the DNA sequence that is complementary to the protospacer sequence; f) comparing the estimated lifetimes at the protospacer and/or off-target sites of the first gRNA with the estimated lifetimes of the full-length gRNA or a truncated gRNA (tru-gRNA) at the protospacer and/or off-target sites; g) randomizing 0 to N nucleotides in the linker and 0 to M nucleotides in the first gRNA and generating a second gRNA and repeating step (e) with the second gRNA; h) identifying an optimized gRNA based on a gRNA sequence that satisfy a design criteria; and i) testing the optimized gRNA in vivo to determine the specificity of binding.

The present invention is directed to a method of generating an optimized guide RNA (gRNA). The method comprises: a) identifying a target region of interest, the target region of interest comprising a protospacer sequence; b) determining a polynucleotide sequence of a full-length gRNA that targets the target region of interest, the full-length gRNA comprising a protospacer-targeting sequence or segment; c) determining at least one or more off-target sites for the full-length gRNA; d) generating a polynucleotide sequence of a first gRNA, the first gRNA comprising the polynucleotide sequence of the full-length gRNA and a RNA segment, the RNA segment comprising a polynucleotide sequence having a length of M nucleotides that is complementary to a nucleotide segment of the protospacer-targeting sequence or segment, the RNA segment is at the 3′ end of the polynucleotide sequence of the full-length gRNA, the first gRNA optionally comprising a linker between the 3′ end of the polynucleotide sequence of the full-length gRNA and the RNA segment, the linker comprising a polynucleotide sequence having a length of N nucleotides, the first gRNA capable of invading the protospacer sequence and binding to a DNA sequence that is complementary to the protospacer sequence and forming a protospacer-duplex, and the first gRNA capable of invading an off-target site and binding to a DNA sequence that is complementary to the off-target site and forming an off-target duplex; e) calculating an estimate or computationally simulating the invasion kinetics and lifetime that the first gRNA remains invaded in the protospacer and off-target site duplexes, wherein the dynamics of invasion are estimated nucleotide-by-nucleotide by determining the energetic differences between further invasion of a different gRNA and re-annealing of the first gRNA to the DNA sequence that is complementary to the protospacer sequence; f) comparing the estimated lifetimes at the protospacer and/or off-target sites of the first gRNA with the estimated lifetimes of the full-length gRNA or a truncated gRNA (tru-gRNA) at the protospacer and/or off-target sites; g) randomizing 0 to N nucleotides in the linker and 0 to M nucleotides in the first gRNA and generating a second gRNA and repeating step (e) with the second gRNA; h) identifying an optimized gRNA based on a gRNA sequence that satisfy a design criteria; and i) testing the optimized gRNA in vivo to determine the specificity of binding.

The present invention is directed to an optimized gRNA generated by the methods described above.

The present invention is directed to an isolated polynucleotide encoding the optimized gRNA described above.

The present invention is directed to a vector comprising the isolated polynucleotide described above.

The present invention is directed to a cell comprising the isolated polynucleotide described above or the vector described above.

The present invention is directed to a kit comprising the isolated polynucleotide described above, the vector described above, or the cell described above.

The present invention is directed to a method of epigenomic editing in a target cell or a subject. The method comprises contacting a cell or a subject with an effective amount of the optimized gRNA molecule described above and a fusion protein, the fusion protein comprising a first polypeptide domain comprising a nuclease-deficient Cas9 and a second polypeptide domain having an activity selected from the group consisting of transcription activation activity, transcription repression activity, nuclease activity, transcription release factor activity, histone modification activity, nucleic acid association activity, DNA methylase activity, and direct or indirect DNA demethylase activity.

The present invention is directed to a method of site specific DNA cleavage in a target cell or a subject. The method comprises contacting a cell or a subject with an effective amount of the optimized gRNA molecule described above and a fusion protein or Cas9 protein, the fusion protein comprising a first polypeptide domain comprising a nuclease-deficient Cas9 and a second polypeptide domain having an activity selected from the group consisting of transcription activation activity, transcription repression activity, nuclease activity, transcription release factor activity, histone modification activity, nucleic acid association activity, DNA methylase activity, and direct or indirect DNA demethylase activity.

The present invention is directed to a method of genome editing in a cell. The method comprises administering to the cell an effective amount of the optimized gRNA molecule described above and a fusion protein, the fusion protein comprising a first polypeptide domain comprising a nuclease-deficient Cas9 and a second polypeptide domain having an activity selected from the group consisting of transcription activation activity, transcription repression activity, nuclease activity, transcription release factor activity, histone modification activity, nucleic acid association activity, DNA methylase activity, and direct or indirect DNA demethylase activity.

The present invention is directed to a method of modulating gene expression in a cell. The method comprises contacting the cell with an effective amount of the optimized gRNA described above and a fusion protein, the fusion protein comprising a first polypeptide domain comprising a nuclease-deficient Cas9 and a second polypeptide domain having an activity selected from the group consisting of transcription activation activity, transcription repression activity, nuclease activity, transcription release factor activity, histone modification activity, nucleic acid association activity, DNA methylase activity, and direct or indirect DNA demethylase activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a schematic representation of Cas9 activity.

FIG. 1B shows an atomic force microscopy (AFM) image of dCas9-sgRNA bound at the protospacer sequence within a single streptavidin-labeled DNA molecule derived from the human AAVS1 locus.

FIGS. 1C-ID show fraction of bound DNA occupied by Cas9/dCas9-sgRNA along an AAVS1-derived (FIG. 1C) or an engineered DNA substrate (FIG. 1D) designed with a series of fully-complementary and partially-complementary protospacer sequences. Vertical lines represent the (23 bp) segments where each significant feature is located on the respective substrates.

FIGS. 2A-2D show modulation of binding affinity and specificity by guide RNA variants. FIG. 2A shows a schematic of dCas9 bound to a single-guide RNA with a two nucleotide truncation from its 5′-end (tru-gRNA, purple). FIG. 2B shows a schematic and proposed mechanism of dCas9 bound to a single-guide RNA with 5′-end extension that forms a hairpin with the PAM-distal binding segment of its targeting region (hp-gRNA, blue). FIG. 2C shows single-site binding affinities (K_(A)) for dCas9 with tru-gRNA (purple, n=257) along the engineered DNA substrate (see FIG. 1D). Dashed line shows the single-site affinities of dCas9-sgRNA for comparison. FIG. 2D shows single-site binding affinities (K_(A)) for dCas9 with guide RNAs with 5′-hairpins that overlap the nucleotides complementary to the last six (hp6-gRNA, blue) or ten (hp10-gRNA, green) PAM-distal nucleotides of the protospacer.

FIGS. 3A-3D show Cas9 undergoes a progressive conformational transition as it binds to sites that increasingly match the protospacer sequence. FIG. 3A shows fraction of bound DNA occupied by Cas9/dCas9 along the DNA substrates, with colours representing populations of Cas9/dCas9 clustered according to their structures (by mean-squared difference after alignment, see text). Different features on DNA that were used for site-specific analysis of Cas9/Cas9 structural properties labelled as: non-specific sequences (a; ‘20 MM’), sites containing 10 PAM-distal mismatches within the protospacer (0, ‘10 MM’), sites containing 5 PAM-distal mismatches within the protospacer (γ, ‘5 MM’), or the full protospacer site (6 or F for dCas9 or Cas9, respectively; ‘0 MM’). The ensemble average of the primary clusters are displayed in FIG. 3C and color-coded according to the clustered structures they represent. FIG. 3B shows volume vs. height of Cas9/dCas9 observed, color-coded by the cluster to which each protein was assigned. Dashed lines delineate regions likely composed of aggregates (top right) or streptavidin labels adsorbed near DNA (bottom left). For comparison-mean height of streptavidin end-labels: 0.92 nm+0.006 nm (SEM); mean volume of streptavidin end-labels: 0.110×10⁴ nm³-0.002×10⁴ nm³ (SEM); n=1941. FIG. 3D shows mean volumes and heights of Cas9/dCas9 with sgRNAs (red circles, with red labels for Cas9 and blue labels for dCas9) or tru-gRNAs (purple circles) bound at each feature on the substrates. Note that dCas9 with tru-gRNAs are only expected to interact the first 3 or 8 PAM-distal mismatches of the 5 MM and 10 MM sites (labelled ‘3 MM’ and ‘8 MM’ here, respectively). For standard errors of mean volumes and heights, see Table 2. For Cas9/dCas9 with sgRNAs, their structural properties at each feature are statistically distinct (δ-ε, α-ε: p<0.05; α-β: p<0.005; β-γ, γ-δ: p<<0.0005. Hotelling's T² test).

FIGS. 4A-4D show Kinetic Monte Carlo (KMC) experiments revealing differences in the stability of the R-loop, or the structure formed by the protospacer duplex with an invading guide RNA, within stably bound Cas9 for different guide RNA variants. FIG. 4A shows a schematic of strand invasion of the protospacer (green) by the guide RNA (red) for KMC experiments. The R-loop is highlighted. Transition rates for invasion (vffor the rate of m→m+1, where m is the extent of the strand invasion or, equivalently, the length of the R-loop) or duplex re-annealing (v_(r) for the rate of m→m−1) are a function of the nearest-neighbour DNA:DNA and RNA:DNA hybridization energies. See text and Supplementary Methods for details. FIG. 4B shows Fractional time that the R-loop is of size m for sg-RNAs (red) or tru-gRNA (purple) derived from KMC experiments ‘at equilibrium’ (simulation initiated at m=20 or 18, respectively). Simulation run until t≥10,000 (arbitrary units). FIG. 4C shows kinetic Monte Carlo time course of the R-loop ‘breathing’ for sgRNA (red) and tru-gRNA (purple) after full invasion (simulation initiated at m=20 or 18, respectively). Asterisks highlight the starting position for the simulation. (insert) Histogram of the respective lifetimes during which the R-loop is ≥16 bp long. FIG. 4D shows proposed model for the mechanisms governing Cas9/dCas9 specificity, based on results of AFM imaging and kinetic Monte Carlo (KMC) experiments (see main text). Cas9/dCas9 binds to the PAM and the guide RNA invades into the PAM-adjacent protospacer duplex. During this strand invasion, the guide RNA must displace the complementary strand of the protospacer. Competition between invasion and re-annealing of the duplex results in a dynamic (‘breathing’) R-loop structure. The stability of the 14^(th)-17^(th) sites of the protospacer-guide RNA interaction, which is dramatically increased by binding at the 19^(th) and 20^(th) sites, promotes a conformational change in the Cas9/dCas9 that authorizes DNA cleavage in Cas9.

FIGS. 5A-5C show Kinetic Monte Carlo (KMC) experiments reveal differences in ability to traverse mismatches (MM) and invade the protospacer depending on guide RNA structure. FIGS. 5A-5B show fractional occupancy by time of R-loop lengths m for sgRNA (FIG. 5A) or tru-gRNA (FIG. 5B) during invasion derived from KMC experiments (initiated at m 10, highlighted by asterisk). White X's indicate positions of mismatches. Simulation run until t≥10,000 (arbitrary units) and the results are averaged over 100 trials. FIG. 5C shows representative KMC time courses for strand invasion (starting at m=10) with a mismatched site at m=14 (arrow) for sgRNA (red) and tru-gRNA (purple). While sgRNAs are largely stably invaded after bypassing a mismatch, tru-gRNAs are repeatedly re-trapped behind the mismatch as a result of the inherent volatility of their R-loops (see FIG. 4 ).

FIGS. 6A-6B show experimental (Hsu et al. (2013) Nature biotechnology, 31, 827-832) cutting frequencies at target sites containing a single rG·dG, rC·dC, rA·dA, and rU·dT mismatch in the PAM-distal region (≥10^(th) protospacer site) are correlated with stabilities of the R-loop determined from kinetic Monte Carlo experiments. FIG. 6A shows log₁₀(p-value) of the correlations between Cas9 cutting frequency and stability of R-loop at sites m (fraction of time the guide RNA remains bound to the protospacer at site m, see text) during strand invasion initiated at site m. (i) Stability at sites m=10 to m=14 is highly anti-correlated with the probability that the guide RNA will fall off the protospacer prior to traversing the mismatch (FIG. 5B), while (ii) sites m=14 to m=17 are associated (from AFM images) with the conformational change which induces cleavage activity. Colour corresponds to the correlation coefficient. FIG. 6B shows experimental cutting frequency does not correlate significantly with estimated guide RNA—protospacer equilibrium binding free energies (ΔG° 37) (left), while it does with stability of site m=14 during strand invasion (right). Error bars are standard errors of the mean occupancy time at site m=14. For these kinetic Monte Carlo experiments, max(t)=100 (arbitrary units). Colour bar is used to show the location of the mismatched (MM) site.

FIGS. 7A-7C show a summary of proposed mechanisms by which the structure of the guide RNA affects Cas9/dCas9 specificity. FIG. 7A shows that for the single guide RNA (sgRNA), the first few nucleotides of the RNA (which bind to the 18th-20th sites of the protospacer) stabilize R-loop breathing and binding at the 14th-17th sites of the protospacer, allow efficient conformational transition to the active state to permit cleavage. However, this increased stability imparted by these bases allows for transient stabilization at mismatched sites and the conformational change permitting cleavage. In many cases, having traversed a mismatch, R-loops remain stably fully-invaded. FIG. 7B shows that for guide RNAs with the first few (here 2) nucleotides truncated (tru-gRNA), the reduced stability of the R-loop (characterized by significant volatility) decreases the probability of maintaining the active conformation. When there are mismatched sites in the protospacer, the volatility of the R-loop ensures that it will becomes quickly and repeatedly ‘re-trapped’ behind the mismatch and greatly hindered at those sites. FIG. 7C shows that while ‘simple’ extensions of the 5′-end of the guide RNA to target the protospacer and adjoining sites beyond the protospacer was found to be digested back to approximately sgRNA length in vivo (FIG. 7A), guide RNAs with 5′-hairpins complementary to ‘PAM-distal’-targeting segments (hp-gRNAs) are anticipated to remain protected within the structure of the Cas9/dCas9 prior to invasion. After binding a PAM site and initiating strand invasion by the hp-gRNA, upon binding to a full protospacer the hairpin is opened and full strand invasion can occur. If there are PAM-distal mismatches at the target site, then it is more energetically favorable for the hairpin to remain closed and strand invasion is hindered. The ability for Cas9-hp-gRNAs to cleave RNA remains to be verified.

FIGS. 8A-8B show purity of expressed Cas9 and dCas9 in SDS gel of purified Cas9 (FIG. 8A) and dCas9 (FIG. 8B) products (nominal molecular weight: 160 kDa). Eluted bands show product is ˜95% pure.

FIGS. 9A-9C show additional images of Cas9/dCas9 bound to DNA. A) Binding distribution of dCas9 to substrate containing no homology to the AAVs1 protospacer sequence (compare with FIG. 1 ) (n=443). Overlaid is the cumulative distribution (CDF) of PAM sites (CDF_(PAM), black) and CDF of bases bound by dCas9 (red, CDF_(Cas9)). Comparison begins 100 bases from each end to avoid artifacts introduced by overlap with streptavidin tag (a criteria for DNA selection) and binding to exposed blunt ends of DNA (resulting in expected increase in non-specific binding). B) Absolute difference D_(n) between CDF of protein binding and of PAM sites. Dashed line is Kolmogorov-Smirnov criterion for goodness-of-fit of two distributions. C) CDF of binding was compared to CDF of PAM distributions from 100,000 randomly generated sequences with same probabilities of G, A, T, and C using MATLAB. Vertical red line is experimental Sup(D_(n)), indicating that experimental dCas9 binding more closely matches the experimental PAM distribution than it does to 71.20% of generated sequences.

FIGS. 10A-10C show binding to ‘nonsense’ substrate containing no homology (>3 bp) to protospacer sequence. (A) Images of dCas9 alone. (B) Histogram (n=423) of volume (left) and height (right) of dCas9 imaged alone with Gaussian fit to primary peaks. From the Gaussian fits: mean height is 1.746 nm (95% confidence: 1.689 nm-1.802 nm) with standard deviation 0.441 nm, and mean volume is 1302 nm³ (95% confidence: 1266 nm³-1337 nm³) with standard deviation 259.1 nm³ (note that because the dCas9 here do not have a DNA within its binding channel, their recorded volumes may appear artificially low because of decreased mechanical resistance to the AFM probe). The heights were measured relative to the median value of a 10-pixel area surrounding each protein, and the volumes recorded as the contiguous features greater than twice the standard deviation of the local background heights. (C) Additional representative images of dCas9 bound to DNA which has been labeled at one end with a monovalent streptavidin.

FIGS. 11A-11D show a representative figure of dCas9-sgRNA bound to RNA and example of processing of protein structural properties. FIG. 11A shows a representative wide-field image of dCas9 bound to engineered DNA. FIG. 11B shows a close-up of boxed region. White arrows are monovalent streptavidin and red arrows are dCas9 proteins. FIGS. 11C-11D show an example of extraction from original image (FIG. 11C) and isolation (FIG. 11D) of Cas9/dCas9 structures. This extraction was repeated for each isolated protein bound to the DNA, then aligned pair-wise through iterative translation, rotation, and reflection to minimize their mean-squared topological difference. From these minimized mean-squared differences a distance matrix was composed, clustered each protein according to the method of Laio and Rodriguez (2014) Science (New York, N.Y.), 344, 1492-1496, then mapped the populations of structures by cluster back to their sites on the DNA (FIG. 2A, FIGS. 10A-10C).

FIGS. 12A-12B show properties of Cas9/dCas9-sgRNAs mapped to their respective binding sites. Upper: Stacked histograms of the volume (left), maximum heights (middle), and structures (clustered by mean squared difference) after alignment (right, see text) for all experimental conditions. Populations are colored according to binned volume, height or structural cluster as in the scatter plot below. The binding distribution of extracted Cas9/dCas9 molecules (FIGS. 10A-10C) closely matches that of the entire dataset (FIG. 1C-1D, FIGS. 8A-8B), indicating that the selection procedure is unbiased and the selected proteins are representative of the whole data set. Lower: Scatter plot of volume vs. maximum height of all Cas9/dCas9 color-coded by binned (left) volume, (middle) maximum height, and (right) structural cluster.

FIG. 13 shows structural properties of Cas9/dCas9 with tru-gRNA and hp-gRNAs at their respective binding sites. Fraction of bound DNA occupied by Cas9/dCas9 with along the engineered DNA substrate, with colors representing populations of Cas9/dCas9 clustered according to their structures (see FIG. 3C). Protein structures were classified according to the dCas9/Cas9 with sgRNA that they most closely resembled (by mean-squared difference after alignment, see text). For reference, on the engineered DNA substrates, location of full protospacer site: 144-167 bp; location of 10 MM (8 MM) site: 452-465 bp; location of 5 MM (3 MM) site: 592-610 bp. Similar trends as was seen with dCas9/Cas9 with sgRNAs were seen: as dCas9 binds to sites which increasingly match the mismatch, the fraction of population clustering with the largest (yellow) group increases, although this effect is depressed in tru-gRNA, with a sizable fraction of the population clustering with smaller (green and blue) populations even at the full protospacer site. The effect for hp10-gRNA is particularly pronounced, emphasizing that it has poor affinities for off-target sites.

FIGS. 14A-14C show model of the strand invasion of DNA protospacers by guide RNAs, and estimated binding stabilities of RNA invaded into protospacers with PAM-distal mismatches. FIG. 14A shows a schematic model of strand invasion of DNA protospacers by guide RNAs. See also FIG. 4A. Guide RNA is presumed to dissociated when m=1. FIG. 14B shows the calculated probability distribution of dissociation times for a guide RNA initially invaded up to m=5 for protospacers with different numbers of contiguous PAM-distal mismatches. The length of these dissociation times can be viewed as an approximation of dCas9 binding propensity at those sites. The asterisk highlights the dissociation times for the population of guide RNAs which initially fails to fully invade after initial invasion to m=5. The invaded RNAs are highly unstable at protospacer sites with 15 PAM-distal mismatches (15 MM), and experimentally we rarely observe Cas9/dCas9 bound at these sites (FIG. 1D). The invaded RNA (prior to dissociation) at protospacer sites with 10 or 5 PAM-distal mismatches (10 MM and 5 MM) are calculated to remain for significantly longer than those at 15 MM sites, but within an order of magnitude of each other; we find their binding propensity to be approximately equal and lower than full protospacer sites (0 MM) in AFM experiments. The probability density functions were calculated using a Q-matrix method as described (Sakmann et al. (1995) Single-channel recording, Springer; 2nd ed.), using the sequence-specific transition rates between the m states (v_(f) and v_(r), see Supplementary Methods). FIG. 14C shows examination of the estimated half-lives of RNA-protospacer binding at protospacers with different numbers of PAM-distal mismatches suggests there are roughly three regimes within which the stabilities of the invaded RNA are similar: those with >11 PAM-distal mismatches (low stability); those with between 3 and 11 PAM-distal mismatches (medium stability); and those with <3 PAM-distal mismatches (high stability). The results are qualitatively similar to the distribution of dCas9 on the engineered substrate observed via AFM (FIG. 1D).

FIG. 15 shows simulated mean first passage times to traverse the mismatched site during strand invasion by sgRNA and tru-gRNA. Simulated (kinetic Monte Carlo) mean first passage times to traverse the mismatched site during strand invasion by sgRNA (blue) and tru-gRNA (red) for different positions of the mismatched site. Error bars are standard deviations of recorded first passage times. Sequence of protospacer (AAVS1 site) in box.

FIGS. 16A-16B shows correlations between Cas9 cleavage frequency (Hsu et al. (2013) Nature Biotechnology, 31, 827-832) and measures of R-loop stability derived from kinetic Monte Carlo. FIG. 16A shows statistical power and strength of the correlations between stability of R-loop sites (from kinetic Monte Carlo, see main text) and experimental cleavage frequency from Hsu et al. (2013) Nature Biotechnology, 31, 827-832 decrease with increasing simulation length (max(t)=100 to max(t)=1000, arbitrary units). This result suggests that the kinetics of strand invasion can be an important predictor of off-target cleavage rate. FIG. 16B shows correlation between fractions of time the R-loop is of size m vs. the probability that the kinetic Monte Carlo trial predicts that the invading strand will dissociate before traversing the mismatch. Binding at sites 10˜14-15 is very strongly anti-correlated (˜0.5-0.85) with the probability of dissociation before traversing the mismatch, while from the AFM imaging experiments we find that binding at sites ˜≥16 are associated with a conformational change in the Cas9/dCas9.

FIG. 17 shows a summary of Deep-Seq data, comparing ontarget activities.

FIG. 18 shows a summary of Deep-Seq data, comparing specificity increases.

FIG. 19 shows protospacer1, Dystrophin; Lane 1 shows GFP Control; Lane 2 shows Full gRNA; Lane 3 shows Tru-gRNA 19 nt; Lane 4 shows Tru-gRNA 18 nt; Lane 5 shows Tru-gRNA 17 nt; Lane 6 shows Tru-gRNA 16 nt; Lane 7 shows Hp-gRNA 4 bp; Lane 8 shows Hp-gRNA 5 bp; Lane 9 shows Hp-gRNA 6 bp; Lane 10 shows Hp-gRNA 7 bp; Lane 11 shows Hp-gRNA 8 bp; and Lane 12 shows Hp-gRNA 9 bp, hairpin1 (Lane 12, 9 nt hp)—GtgagtaggttcgCCTACTCAGACTGTTACTC (SEQ ID NO: 335), wherein italicized is part of hairpin and underlined is the hairpin loop.

FIG. 20 shows protospacer1, Dystrophin, internal loops

FIGS. 21A and 21B show Calculated secondary structures of the 5′-ends of the protospacer-targeting segments of hp-gRNAs used for Deep Seq experiments (using NuPack software suite). Colors are probability of each nucleotide existing in that secondary structure at equilibrium.

FIG. 22 shows Dystrophin, indel rates, all sites

FIG. 23 shows Dystrophin, ontarget/sum(offtargets).

FIG. 24 shows protospacer2, EMX1; Lane 1 shows GFP Control; Lane 2 shows Full gRNA; Lane 3 shows Tru-gRNA; Lane 4 shows 10-bp hp-gRNA; and Lane 5 shows 6-bp hp-gRNA, hairpin1. Conversions—Surv_OT1=DS_OT2; Surv_OT53=DS_OT3.

FIGS. 25A and 25B show protospacer2, EMX1, tru-hps, internal loops.

FIGS. 26A-26C show hairpin structures. FIG. 26A shows hairpin 1 which is a 6 bp 5′-hairpin. FIG. 26B shows hairpin 2 which is a 5 bp 5′-hairpin on 18 nt (truncated) gRNA. FIG. 26C shows hairpin 3 which is a 3 bp 5′-hairpin.

FIG. 27 shows EMX1, Indel rates, all sites.

FIG. 28 shows EMX1, indel rates, low-rate offtargets.

FIG. 29 shows EMX1, ontarget/sum(offtargets).

FIG. 30 shows protospacer3, VEGFA1. Lane 1 shows GFP Control; Lane 2 shows Full gRNA; Lane 3 shows Tru-gRNA; Lane 4 shows 10-bp hp-gRNA; and Lane 5 shows 6-bp hp-gRNA.

FIGS. 31A and 31B show protospacer3, VEGFA1: pam proximal hairpins. Lane 1 shows GFP control; Lane 2 shows Full gRNA; Lane 3 shows hp-gRNA1; Lane 4 shows hp-gRNA2; Lane 5 shows hp-gRNA3; Lane 6 shows hp-gRNA4; Lane 7 shows hp-gRNA5; and Lane 8 shows hp-gRNA6.

FIG. 32 shows protospacer3, VEGFA1: pam proximal hairpins.

FIG. 33 shows protospacer3, VEGF1, internal loops. Lane 1 shows Control; lane 2 shows Full; lane 3 shows 2 nt hp; lane 4 shows 3 nt hp, hairpin 5; and lane 5 shows 4 nt hp.

FIGS. 34A and 34B show Deep-seq Experiments for hairpins 1, 2, and 3 failed. FIG. 25A shows Hairpin 4—Computationally-derived hairpin designed to discriminate against Off-target site 2 while maintaining on-target activity. FIG. 25B shows Hairpin 5-4 bp 5′-hairpin (gRNA normally has significant 3′ secondary structure).

FIG. 35 shows VEGF1, indel rates, all sites.

FIG. 36 shows VEGF1, indel rates, low-rate offtargets.

FIG. 37 shows VEGF1, ontarget/sum(offtargets).

FIG. 38 shows protospacer 4, VEGFA3. Lane 1 shows GFP Control; Lane 2 shows Full gRNA, Lane 3 shows Tru-gRNA; Lane 4 shows 3-bp hp-gRNA; Lane 5 shows 4-bp hp-gRNA; Lane 6 shows 5-bp hp-gRNA; Lane 7 shows 6-bp hp-gRNA; and Lane 8 shows 10-bp hp-gRNA.

FIGS. 39A and 39B show gRNA4, VEGFA3: pam proximal hairpins. Lane 1 shows GFP control; Lane 2 shows Full gRNA; Lane 3 shows hp-gRNA1; Lane 4 shows hp-gRNA2; Lane 5 shows hp-gRNA3; Lane 6 shows hp-gRNA4; Lane 7 shows hp-gRNA5; and Lane 8 shows hp-gRNA6.

FIG. 40A shows Hairpin 1-4 bp hairpin targeting 3′-region.

FIG. 40B shows Hairpin 2-4 bp hairpin targeting 3′-region with G-U wobble pairs.

FIG. 40C shows Hairpin 3-4 bp hairpin targeting 3′-region with G-U wobble pair (variant design).

FIG. 41 shows VEGF3, indel rates, all sites.

FIG. 42 shows VEGF3, indel rates, low-rate offtargets.

FIG. 43 shows VEGF3, ontarget/sum(offtargets).

FIG. 44A shows a hairpin designed to target EMX1 gene.

FIG. 44B shows the EMX1-sg1 sequence of the hairpin of FIG. 44A.

FIG. 44C shows the effect of decreasing protospacer length and increasing hairpin length on specificity.

FIGS. 45A-45D show DNA/RNA Sequences.

FIG. 46 shows a figure that describes the Surveyor assays.

FIG. 47 shows tolerance of AsCpf1 and LbCpf1 to mismatched or truncated crRNAs and endogenous gene modification by AsCpf1 and LbCpf1 using crRNAs that contain singly mismatched bases. Activity determined by T7E1 assay; error bars, s.e.m.; n=3 (taken from Kleinstiver et al., Nat. Biotech. 34:869-875).

FIG. 48 shows surveyor assay results for hp-gRNAs used with a Type V CRISPR system in which a hairpin is added to the 3′ end of a full-length gRNA to abolish off-target activity.

DETAILED DESCRIPTION

Disclosed herein are composition and methods for site specific DNA targeting and epigenomic gene editing and/or transcriptional regulation, such as DNA cleavage and gene activation or repression. The present invention is directed to a modular method for designing and using optimized guide RNAs that have hairpin structures (hpgRNA) that can be easily incorporated into the existing biotechnology infrastructure and which results in a controlled decrease of off-target activity, all while maintaining the ability to target the correct DNA sequence specifically. The methods described herein provide a novel approach to engineering the optimized gRNA to perform significantly better than other available methods and can be used in combination with other protein-specific means of improving increasing specifically for highly improved performance.

The disclosed methods and optimized gRNAs have the great advantage of being easily adapted to current methodologies and infrastructures already in place to perform RNA-guided genomic engineering. In some embodiments, Cas9, dCas9, or Cpf1 are delivered into a cell using viral vectors along with vectors coding for the transcription of the optimized gRNAs in the cell. The current invention would require only a few additional nucleotides to the vector coding for the optimized gRNA, which can be easily accommodated by the current and standard practices. Like truncated guide RNAs (tru-gRNAs), the optimized gRNAs or hpgRNAs can be used in combination with paired nickases, for example, or other modifications of the endonucleases themselves to further improve specificity. A series of experiments were performed in vitro which showed that the use of the optimized gRNAs produced using the methods described herein increased the specificity in DNA binding relative to the best available gRNA options (see FIG. 2 ). The use of the optimized gRNA abolishes or significantly weakens activity at targets containing only a few mismatched DNA sequences, which tend to be the sites at which off-target activity by RNA-guided endonucleases occurs. The optimized gRNA also provide specificity of cleavage activity in mammalian cells at sites which are known to induce off-target activity even in the best known improvements to the guide RNAs. The invention is a generally-applicable method to decrease off-target activity by RNA-guided endonucleases, particularly Cas9, by engineering changes the structural design of the guide RNA.

1. DEFINITIONS

The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

“Adeno-associated virus” or “AAV” as used interchangeably herein refers to a small virus belonging to the genus Dependovirus of the Parvoviridae family that infects humans and some other primate species. AAV is not currently known to cause disease and consequently the virus causes a very mild immune response.

“Binding region” as used herein refers to the region within a nuclease target region that is recognized and bound by the nuclease, such as Cas9.

“Chromatin” as used herein refers to an organized complex of chromosomal DNA associated with histones.

“Cis-regulatory elements” or “CREs” as used interchangeably herein refers to regions of non-coding DNA which regulate the transcription of nearby genes. CREs are found in the vicinity of the gene, or genes, they regulate. CREs typically regulate gene transcription by functioning as binding sites for transcription factors. Examples of CREs include promoters, enhancers, super-enhancers, silencers, insulators, and locus control regions.

“Clustered Regularly Interspaced Short Palindromic Repeats” and “CRISPRs”, as used interchangeably herein refers to loci containing multiple short direct repeats that are found in the genomes of approximately 40% of sequenced bacteria and 90% of sequenced archaea.

“Coding sequence” or “encoding nucleic acid” as used herein means the nucleic acids (RNA or DNA molecule) that comprise a nucleotide sequence which encodes a protein. The coding sequence can further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may be codon optimize.

“Complement” or “complementary” as used herein means a nucleic acid can mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules. “Complementarity” refers to a property shared between two nucleic acid sequences, such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary.

“Correcting”, “genome editing” and “restoring” as used herein refers to changing a mutant gene that encodes a truncated protein or no protein at all, such that a full-length functional or partially full-length functional protein expression is obtained. Correcting or restoring a mutant gene may include replacing the region of the gene that has the mutation or replacing the entire mutant gene with a copy of the gene that does not have the mutation with a repair mechanism such as homology-directed repair (HDR). Correcting or restoring a mutant gene may also include repairing a frameshift mutation that causes a premature stop codon, an aberrant splice acceptor site or an aberrant splice donor site, by generating a double stranded break in the gene that is then repaired using non-homologous end joining (NHEJ). NHEJ may add or delete at least one base pair during repair which may restore the proper reading frame and eliminate the premature stop codon. Correcting or restoring a mutant gene may also include disrupting an aberrant splice acceptor site or splice donor sequence. Correcting or restoring a mutant gene may also include deleting a non-essential gene segment by the simultaneous action of two nucleases on the same DNA strand in order to restore the proper reading frame by removing the DNA between the two nuclease target sites and repairing the DNA break by NHEJ.

“Demethylases” as used herein refers to an enzyme that removes methy (CH3-) groups from nucleic acids, proteins (in particular histones), and other molecules. Demethylase enzymes are important in epigenetic modification mechanisms. The demethylase proteins alter transcriptional regulation of the genome by controlling the methylation levels that occur on DNA and histones and, in turn, regulate the chromatin state at specific gene loci within organisms. “Histone demethylase” refers to a methylase that removes methy groups from histones. There are several families of histone demethylases, which act on different substrates and play different roles in cellular function. The Fe(II)-dependent lysine demethylases may be a JMJC demethylase. A JMJC demethylase is a histone demethylase containing a JumonjiC (JmjC) domain. The JMJC demethylase may be a member of the KDM3, KDM4, KDM5, or KDM6 family of histone demethylases.

“DNase I hypersensitive sites” or “DHS” as used interchangeably herein refers to docking sites for the transcription factors and chromatin modifiers, including p300 that coordinate distal target gene expression.

“Donor DNA”, “donor template” and “repair template” as used interchangeably herein refers to a double-stranded DNA fragment or molecule that includes at least a portion of the gene of interest. The donor DNA may encode a full-functional protein or a partially-functional protein.

“Endogenous gene” as used herein refers to a gene that originates from within an organism, tissue, or cell. An endogenous gene is native to a cell, which is in its normal genomic and chromatin context, and which is not heterologous to the cell. Such cellular genes include, e.g., animal genes, plant genes, bacterial genes, protozoal genes, fungal genes, mitochondrial genes, and chloroplastic genes. An “endogenous target gene” as used herein refers to an endogenous gene that is targeted by an optimized gRNA and CRISPR/Cas9-based system or CRISPR/Cpf1-based system.

“Enhancer” as used herein refers to non-coding DNA sequences containing multiple activator and repressor binding sites. Enhancers range from 50 bp to 1500 bp in length and may be either proximal, 5′ upstream to the promoter, within any intron of the regulated gene, or distal, in introns of neighboring genes, or intergenic regions far away from the locus, or on regions on different chromosomes. More than one enhancer may interact with a promoter. Similarly, enhancers may regulate more than one gene without linkage restriction and may “skip” neighboring genes to regulate more distant ones. Transcriptional regulation may involve elements located in a chromosome different to one where the promoter resides. Proximal enhancers or promoters of neighboring genes may serve as platforms to recruit more distal elements.

“Duchenne Muscular Dystrophy” or “DMD” as used interchangeably herein refers to a recessive, fatal, X-linked disorder that results in muscle degeneration and eventual death. DMD is a common hereditary monogenic disease and occurs in 1 in 3500 males. DMD is the result of inherited or spontaneous mutations that cause nonsense or frame shift mutations in the dystrophin gene. The majority of dystrophin mutations that cause DMD are deletions of exons that disrupt the reading frame and cause premature translation termination in the dystrophin gene. DMD patients typically lose the ability to physically support themselves during childhood, become progressively weaker during the teenage years, and die in their twenties.

“Dystrophin” as used herein refers to a rod-shaped cytoplasmic protein which is a part of a protein complex that connects the cytoskeleton of a muscle fiber to the surrounding extracellular matrix through the cell membrane. Dystrophin provides structural stability to the dystroglycan complex of the cell membrane that is responsible for regulating muscle cell integrity and function. The dystrophin gene or “DMD gene” as used interchangeably herein is 2.2 megabases at locus Xp21. The primary transcription measures about 2,400 kb with the mature mRNA being about 14 kb. 79 exons code for the protein which is over 3500 amino acids.

“Exon 51” as used herein refers to the 51^(st) exon of the dystrophin gene. Exon 51 is frequently adjacent to frame-disrupting deletions in DMD patients and has been targeted in clinical trials for oligonucleotide-based exon skipping. A clinical trial for the exon 51 skipping compound eteplirsen recently reported a significant functional benefit across 48 weeks, with an average of 47% dystrophin positive fibers compared to baseline. Mutations in exon 51 are ideally suited for permanent correction by NHEJ-based genome editing.

“Frameshift” or “frameshift mutation” as used interchangeably herein refers to a type of gene mutation wherein the addition or deletion of one or more nucleotides causes a shift in the reading frame of the codons in the mRNA. The shift in reading frame may lead to the alteration in the amino acid sequence at protein translation, such as a missense mutation or a premature stop codon.

“Full-length gRNA” or “standard gRNA” as used interchangeably herein refers to a gRNA that includes a “scaffold” and a protospacer-targeting sequence or segment that is typically 20 nucleotides in length.

“Functional” and “full-functional” as used herein describes protein that has biological activity. A “functional gene” refers to a gene transcribed to mRNA, which is translated to a functional protein.

“Fusion protein” as used herein refers to a chimeric protein created through the joining of two or more genes that originally coded for separate proteins. The translation of the fusion gene results in a single polypeptide with functional properties derived from each of the original proteins.

“Genetic construct” as used herein refers to the DNA or RNA molecules that comprise a nucleotide sequence that encodes a protein. The coding sequence includes initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered. As used herein, the term “expressible form” refers to gene constructs that contain the necessary regulatory elements operable linked to a coding sequence that encodes a protein such that when present in the cell of the individual, the coding sequence will be expressed.

“Genetic disease” as used herein refers to a disease, partially or completely, directly or indirectly, caused by one or more abnormalities in the genome, especially a condition that is present from birth. The abnormality may be a mutation, an insertion or a deletion. The abnormality may affect the coding sequence of the gene or its regulatory sequence. The genetic disease may be, but not limited to DMD, hemophilia, cystic fibrosis, Huntington's chorea, familial hypercholesterolemia (LDL receptor defect), hepatoblastoma, Wilson's disease, congenital hepatic porphyria, inherited disorders of hepatic metabolism, Lesch Nyhan syndrome, sickle cell anemia, thalassaemias, xeroderma pigmentosum, Fanconi's anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom's syndrome, retinoblastoma, and Tay-Sachs disease.

“Genome” as used herein refers to the complete set of genes or genetic material present in a cell or organism. The genome includes DNA or RNA in RNA viruses. The genome includes both the genes, (the coding regions), the noncoding DNA and the genomes of the mitochondria and chloroplasts.

“guide RNA,” “gRNA,” “single gRNA,” and “sgRNA” as used interchangeably herein refer to a short synthetic RNA composed of a “scaffold” sequence necessary for Cas9-binding or Cpf1-binding and a user-defined “spacer” or “targeting sequence” (also referred to herein as a protospacer-targeting sequence or segment) which defines the genomic target to be modified. “hpgRNA,” “hp-gRNA,” and “optimized gRNA” as used interchangeably herein refer to a gRNA that has additional nucleotides at either the 5′-end or 3′-end that can form a secondary structure with all or part of the protospacer-targeting sequence or segment.

“Histone acetyltransferases” or “HATs” are used interchangeably herein refers to enzymes that acetylate conserved lysine amino acids on histone proteins by transferring an acetyl group from acetyl CoA to form ε-N-acetyllysine. DNA is wrapped around histones, and, by transferring an acetyl group to the histones, genes can be turned on and off. In general, histone acetylation increases gene expression as it is linked to transcriptional activation and associated with euchromatin. Histone acetyltransferases can also acetylate non-histone proteins, such as nuclear receptors and other transcription factors to facilitate gene expression.

“Histone deacetylases” or “HDACs” as used interchangeably herein refers to a class of enzymes that remove acetyl groups (O═C—CH₃) from an ε-N-acetyl lysine amino acid on a histone, allowing the histones to wrap the DNA more tightly. HDACs are also called lysine deacetylases (KDAC), to describe their function rather than their target, which also includes non-histone proteins.

“Histone methyltransferase” or “HMTs” as used interchangeably herein refers to histone-modifying enzymes (e.g., histone-lysine N-methyltransferases and histone-arginine N-methyltransferases), that catalyze the transfer of one, two, or three methyl groups tolysine and arginine residues of histone proteins. The attachment of methyl groups occurs predominantly at specific lysine or arginine residues on histones H3 and H4.

“Homology-directed repair” or “HDR” as used interchangeably herein refers to a mechanism in cells to repair double strand DNA lesions when a homologous piece of DNA is present in the nucleus, mostly in G2 and S phase of the cell cycle. HDR uses a donor DNA template to guide repair and may be used to create specific sequence changes to the genome, including the targeted addition of whole genes. If a donor template is provided along with the site specific nuclease, such as with a CRISPR/Cas9-based system or CRISPR/Cpf1-based system, then the cellular machinery will repair the break by homologous recombination, which is enhanced several orders of magnitude in the presence of DNA cleavage. When the homologous DNA piece is absent, non-homologous end joining may take place instead.

“Genome” as used herein refers to the complete set of genes or genetic material present in a cell or organism. The genome includes DNA or RNA in RNA viruses. The genome includes both the genes, (the coding regions), the noncoding DNA and the genomes of the mitochondria and chloroplasts.

“Genome editing” as used herein refers to changing a gene. Genome editing may include correcting or restoring a mutant gene. Genome editing may include knocking out a gene, such as a mutant gene or a normal gene. Genome editing may be used to treat disease or enhance muscle repair by changing the gene of interest.

“Identical” or “identity” as used herein in the context of two or more nucleic acids or polypeptide sequences means that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity may be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

“Insulators” as used herein refers to a genetic boundary element that blocks the interaction between enhancers and promoters. By residing between the enhancer and promoter, the insulator may inhibit their subsequent interactions. Insulators can determine the set of genes an enhancer can influence. Insulators are needed where two adjacent genes on a chromosome have very different transcription patterns and the inducing or repressing mechanisms of one does not interfere with the neighboring gene. Insulators have also been found to cluster at the boundaries of topological association domains (TADs) and may have a role in partitioning the genome into “chromosome neighborhoods”—genomic regions within which regulation occurs. Insulator activity is thought to occur primarily through the 3D structure of DNA mediated by proteins including CTCF. Insulators are likely to function through multiple mechanisms. Many enhancers form DNA loops that put them in close physical proximity to promoter regions during transcriptional activation. Insulators may promote the formation of DNA loops that prevent the promoter-enhancer loops from forming. Barrier insulators may prevent the spread of heterochromatin from a silenced gene to an actively transcribed gene.

“Invasion” as used herein refers to the disruption of a DNA duplex at a protospacer region in a target region of a target gene, such as by a gRNA that binds to the DNA sequence that is complementary to the protospacer.

“Invasion kinetics” as used herein refers to the rate at which invasion proceeds. Invasion kinetics can refer to the rate at which the guide RNA invades the duplex, either to “full invasion” such that the protospacer is completely invaded, or the rate at which the segment of protospacer DNA bound to the guide RNA expands as it is displaced from its complementary strand and bound to the guide RNA nucleotide-by-nucleotide from its PAM-proximal region through to full invasion.

“Lifetime” as used herein refers to period of time that a gRNA remains invaded in the region in a target region of a target gene.

“Locus control regions” as used herein refers to a long-range cis-regulatory element that enhances expression of linked genes at distal chromatin sites. It functions in a copy number-dependent manner and is tissue-specific, as seen in the selective expression of β-globin genes in erythroid cells. Expression levels of genes can be modified by the LCR and gene-proximal elements, such as promoters, enhancers, and silencers. The LCR functions by recruiting chromatin-modifying, coactivator, and transcription complexes. Its sequence is conserved in many vertebrates, and conservation of specific sites may suggest importance in function.

“Mismatched” or “MM” as used interchangeably herein refers to mismatched bases that include a G/T or A/C pairing. Mismatches are commonly due to tautomerization of bases during G2. The damage is repaired by recognition of the deformity caused by the mismatch, determining the template and non-template strand, and excising the wrongly incorporated base and replacing it with the correct nucleotide.

“Modulate” as used herein may mean any altering of activity, such as regulate, down regulate, upregulate, reduce, inhibit, increase, decrease, deactivate, or activate.

“Mutant gene” or “mutated gene” as used interchangeably herein refers to a gene that has undergone a detectable mutation. A mutant gene has undergone a change, such as the loss, gain, or exchange of genetic material, which affects the normal transmission and expression of the gene. A “disrupted gene” as used herein refers to a mutant gene that has a mutation that causes a premature stop codon. The disrupted gene product is truncated relative to a full-length undisrupted gene product.

“Non-homologous end joining (NHEJ) pathway” as used herein refers to a pathway that repairs double-strand breaks in DNA by directly ligating the break ends without the need for a homologous template. The template-independent re-ligation of DNA ends by NHEJ is a stochastic, error-prone repair process that introduces random micro-insertions and micro-deletions (indels) at the DNA breakpoint. This method may be used to intentionally disrupt, delete, or alter the reading frame of targeted gene sequences. NHEJ typically uses short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the end of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately, yet imprecise repair leading to loss of nucleotides may also occur, but is much more common when the overhangs are not compatible.

“Normal gene” as used herein refers to a gene that has not undergone a change, such as a loss, gain, or exchange of genetic material. The normal gene undergoes normal gene transmission and gene expression.

“Nuclease mediated NHEJ” as used herein refers to NHEJ that is initiated after a nuclease, such as a cas9, cuts double stranded DNA.

“Nucleic acid” or “oligonucleotide” or “polynucleotide” as used herein means at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. A single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.

Nucleic acids may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods.

“On-target site” as used herein refers to the target region or sequence in a genome to which the gRNA is intended to target. Ideally, the on-target site has perfect homology (100% identity or homology) to the target DNA sequence with no homology elsewhere in the genome.

“Off-target site” as used herein refers to a region of the genome which has partial homology or partial identity to the on-target site or target region of the gRNA, but which the gRNA is not intended or designed to target.

“Operably linked” as used herein means that expression of a gene is under the control of a promoter with which it is spatially connected. A promoter may be positioned 5′ (upstream) or 3′ (downstream) of a gene under its control. The distance between the promoter and a gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance may be accommodated without loss of promoter function.

“p300 protein,” “EP300,” or “E1A binding protein p300” as used interchangeably herein refers to the adenovirus E1A-associated cellular p300 transcriptional co-activator protein encoded by the EP300 gene. p300 is a highly conserved acetyltransferase involved in a wide range of cellular processes ENREF 30. p300 functions as a histone acetyltransferase that regulates transcription via chromatin remodeling and is involved with the processes of cell proliferation and cell differentiation.

“Partially-functional” as used herein describes a protein that is encoded by a mutant gene and has less biological activity than a functional protein but more than a non-functional protein.

“Premature stop codon” or “out-of-frame stop codon” as used interchangeably herein refers to nonsense mutation in a sequence of DNA, which results in a stop codon at location not normally found in the wild-type gene. A premature stop codon may cause a protein to be truncated or shorter compared to the full-length version of the protein.

“Primary cell” as used herein refers to cells taken directly from living tissue (e.g. biopsy material). Primary cells can be established for growth in vitro. These cells have undergone very few population doublings and are therefore more representative of the main functional component of the tissue from which they are derived in comparison to continuous (tumor or artificially immortalized) cell lines thus representing a more representative model to the in vivo state. Primary cells may be taken from different species, such as mouse or humans.

“Protospacer sequence” or “protospacer segment” as used interchangeably herein refers to a DNA sequence targeted by the Cas9 nuclease or Cpf1 nuclease in the CRISPR bacterial adaptive immune system. In the CRISPR/Cas9 system, the protospacer sequence is typically followed by a protospacer-adjacent motif (PAM); the PAM is at the 5′-end. In the CRISPR/Cpf1 system, PAM is followed by the protospacer sequence; the PAM is at the 3′-end.

“Protospacer-targeting sequence” or “protospacer-targeting segment” as used interchangeably herein refers to a nucleotide sequence of a gRNA that corresponds to the protospacer sequence and facilitates targeting of the CRISPR/Cas9-based system or CRISPR/Cpf1-based system to the protospacer sequence.

“Promoter” as used herein means a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of same. A promoter may also comprise distal enhancer or repressor elements, which may be located as much as several thousand base pairs, or anywhere in the genome, from the start site of transcription. A promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter may regulate the expression of a gene component constitutively, or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, hormones, toxins, drugs, pathogens, metal ions, or inducing agents. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter and the CMV IE promoter.

“Protospacer adjacent motif” or “PAM” as used herein refers to a DNA sequence immediately following the DNA sequence targeted by the Cas9 or immediately before the DNA sequence targeted by the Cpf1 nuclease in the CRISPR bacterial adaptive immune system. PAM is a component of the invading virus or plasmid, but is not a component of the bacterial CRISPR locus. Cas9 and Cpf1 will not successfully bind to or cleave the target DNA sequence if it is not followed by or preceded by the PAM sequence, respectively. PAM is an essential targeting component (not found in bacterial genome) which distinguishes bacterial self from non-self DNA, thereby preventing the CRISPR locus from being targeted and destroyed by nuclease.

The term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (naturally occurring) form of the cell or express a second copy of a native gene that is otherwise normally or abnormally expressed, under expressed or not expressed at all.

“Silencers” or “repressors” as used interchangeably herein refer to a DNA sequence capable of binding transcription regulation factors and preventing genes from being expressed as proteins. A silencer is a sequence-specific element that induces a negative effect on the transcription of its particular gene. There are many positions in which a silencer element can be located in DNA. The most common position is found upstream of the target gene where it can help repress the transcription of the gene. This distance can vary greatly between approximately −20 bp to −2000 bp upstream of a gene. Certain silencers can be found downstream of a promoter located within the intron or exon of the gene itself. Silencers have also been found within the 3 prime untranslated region (3′ UTR) of mRNA. There are two main types of silencers in DNA, which are the classical silencer element and the non-classical negative regulatory element (NRE). In classical silencers, the gene is actively repressed by the silencer element, mostly by interfering with general transcription factor (GTF) assembly. NREs passively repress the gene, usually by inhibiting other elements that are upstream of the gene.

“Skeletal muscle” as used herein refers to a type of striated muscle, which is under the control of the somatic nervous system and attached to bones by bundles of collagen fibers known as tendons. Skeletal muscle is made up of individual components known as myocytes, or “muscle cells”, sometimes colloquially called “muscle fibers.” Myocytes are formed from the fusion of developmental myoblasts (a type of embryonic progenitor cell that gives rise to a muscle cell) in a process known as myogenesis. These long, cylindrical, multinucleated cells are also called myofibers.

“Skeletal muscle condition” as used herein refers to a condition related to the skeletal muscle, such as muscular dystrophies, aging, muscle degeneration, wound healing, and muscle weakness or atrophy.

“Subject” and “patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgous or rhesus monkey, chimpanzee, etc.) and a human). In some embodiments, the subject may be a human or a non-human. The subject or patient may be undergoing other forms of treatment.

“Super enhancer” as used herein refers to a region of the mammalian genome comprising multiple enhancers that is collectively bound by an array of transcription factor proteins to drive transcription of genes involved in cell identity. Super-enhancers are frequently identified near genes important for controlling and defining cell identity and can be used to quickly identify key nodes regulating cell identity. Enhancers have several quantifiable traits that have a range of values, and these traits are generally elevated at super-enhancers. Super-enhancers are bound by higher levels of transcription-regulating proteins and are associated with genes that are more highly expressed. Expression of genes associated with super-enhancers is particularly sensitive to perturbations, which may facilitate cell state transitions or explain sensitivity of super-enhancer—associated genes to small molecules that target transcription.

“Target enhancer” as used herein refers to enhancer that is targeted by a gRNA and CRISPR/Cas9-based system. The target enhancer may be within the target region.

“Target gene” as used herein refers to any nucleotide sequence encoding a known or putative gene product. The target gene may be a mutated gene involved in a genetic disease.

The “target region”, “target sequence,” “protospacer,” or “protospacer sequence” as used interchangeably herein refers to the region of the target gene to which the CRISPR/Cas9-based system or CRISPR/Cpf1-based system targets.

“Transcribed region” as used herein refers to the region of DNA that is transcribed into single-stranded RNA molecule, known as messenger RNA, resulting in the transfer of genetic information from the DNA molecule to the messenger RNA. During transcription, RNA polymerase reads the template strand in the 3′ to 5′ direction and synthesizes the RNA from 5′ to 3′. The mRNA sequence is complementary to the DNA strand.

“Target regulatory element” as used herein refers to a regulatory element that is targeted by a gRNA and CRISPR/Cas9-based system. The target regulatory element may be within the target region.

“Transcribed region” as used herein refers to the region of DNA that is transcribed into single-stranded RNA molecule, known as messenger RNA, resulting in the transfer of genetic information from the DNA molecule to the messenger RNA. During transcription, RNA polymerase reads the template strand in the 3′ to 5′ direction and synthesizes the RNA from 5′ to 3′. The mRNA sequence is complementary to the DNA strand.

“Transcriptional Start Site” or “TSS” as used interchangeably herein refers to the first nucleotide of a transcribed DNA sequence where RNA polymerase begins synthesizing the RNA transcript.

“Transgene” as used herein refers to a gene or genetic material containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may retain the ability to produce RNA or protein in the transgenic organism, or it may alter the normal function of the transgenic organism's genetic code. The introduction of a transgene has the potential to change the phenotype of an organism.

“tru gRNA” as used herein refers to a full-length guide RNA with nucleotides truncated from their 5′-end, typically 2 nucleotides.

“Trans-regulatory elements” as used herein refers to regions of non-coding DNA which regulate the transcription of genes distant from the gene from which they were transcribed. Trans-regulatory elements may be on the same or different chromosome from the target gene. Examples of trans-regulatory elements include enhancers, super-enhancers, silencers, insulators, and locus control regions.

“Variant” used herein with respect to a nucleic acid means (i) a portion or fragment of a referenced nucleotide sequence (including nucleotide sequences that have insertions or deletions as compared to the referenced nucleotide sequences); (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto.

“Variant” with respect to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Variant may also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes may be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes may be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of 2 are substituted. The hydrophilicity of amino acids may also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide. Substitutions may be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.

“Vector” as used herein means a nucleic acid sequence containing an origin of replication. A vector may be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be a self-replicating extrachromosomal vector, and preferably, is a DNA plasmid. For example, the vector may encode Cas9 and at least one optimized gRNA nucleotide sequence of any one of SEQ ID NOs: 149-315, 321-323, and 326-329.

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

2. CRISPR SYSTEM

The CRISPR system is a microbial nuclease system involved in defense against invading phages and plasmids that provides a form of acquired immunity. The CRISPR loci in microbial hosts can contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage. Short segments of foreign DNA, called spacers, are incorporated into the genome between CRISPR repeats, and serve as a ‘memory’ of past exposures. Cas9 forms a complex with the 3′ end of the single guide RNA (“sgRNA”), and the protein-RNA pair recognizes its genomic target by complementary base pairing between the 5′ end of the sgRNA sequence and a predefined 20 bp DNA sequence, known as the protospacer. This complex is directed to homologous loci of pathogen DNA via regions encoded within the CRISPR RNA (“crRNA”), i.e., the protospacers, and protospacer-adjacent motifs (PAMs) within the pathogen genome. The non-coding CRISPR array is transcribed and cleaved within direct repeats into short crRNAs containing individual spacer sequences, which direct Cas nucleases to the target site (protospacer). By simply exchanging the 20 bp recognition sequence of the expressed chimeric sgRNA, the Cas9 nuclease can be directed to new genomic targets. CRISPR spacers are used to recognize and silence exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms.

Three classes of CRISPR systems (Types I, II and III effector systems) are known. The Type II effector system carries out targeted DNA double-strand break in four sequential steps, using a single effector enzyme, Cas9, to cleave dsDNA. Compared to the Type I and Type III effector systems, which require multiple distinct effectors acting as a complex, the Type II effector system may function in alternative contexts such as eukaryotic cells. The Type II effector system consists of a long pre-crRNA, which is transcribed from the spacer-containing CRISPR locus, the Cas9 protein, and a tracrRNA, which is involved in pre-crRNA processing. The tracrRNAs hybridize to the repeat regions separating the spacers of the pre-crRNA, thus initiating dsRNA cleavage by endogenous RNase III. This cleavage is followed by a second cleavage event within each spacer by Cas9, producing mature crRNAs that remain associated with the tracrRNA and Cas9, forming a Cas9:crRNA-tracrRNA complex.

An engineered form of the Type II effector system of Streptococcus pyogenes was shown to function in human cells for genome engineering. In this system, the Cas9 protein was directed to genomic target sites by a synthetically reconstituted “guide RNA” (“gRNA”, also used interchangeably herein as a chimeric sgRNA, which for Cas9 is a crRNA-tracrRNA fusion that obviates the need for RNase III and crRNA processing in general.

The Cas9:crRNA-tracrRNA complex unwinds the DNA duplex and searches for sequences matching the crRNA to cleave. Target recognition occurs upon detection of complementarity between a “protospacer” sequence in the target DNA and the remaining spacer sequence in the crRNA. Cas9 mediates cleavage of target DNA if a correct protospacer-adjacent motif (PAM) is also present at the 3′ end of the protospacer. For protospacer targeting, the sequence must be immediately followed by the protospacer-adjacent motif (PAM), a short sequence recognized by the Cas9 nuclease that is required for DNA cleavage. Different Type II systems have differing PAM requirements. The S. pyogenes CRISPR system may have the PAM sequence for this Cas9 (SpCas9) as 5′-NRG-3′, where R is either A or G, and characterized the specificity of this system in human cells. A unique capability of the CRISPR/Cas9-based system is the straightforward ability to simultaneously target multiple distinct genomic loci by co-expressing a single Cas9 protein with two or more sgRNAs. For example, the Streptococcus pyogenes Type II system naturally prefers to use an “NGG” sequence, where “N” can be any nucleotide, but also accepts other PAM sequences, such as “NAG” in engineered systems (Hsu et al. (2013) Nature Biotechnology, 31, 827-832). Similarly, the Cas9 derived from Neisseria meningitidis (NmCas9) normally has a native PAM of NNNNGATT, but has activity across a variety of PAMs, including a highly degenerate NNNNGNNN PAM (Esvelt et al. Nature Methods (2013) doi:10.1038/nmeth.2681).

3. CRISPR/CAS9-BASED SYSTEM

Provided herein are CRISPR/Cas9 systems that include an optimized gRNA, such as a hairpin gRNA (also referred herein as “hpgRNA” or “hp-gRNA”), that allow improved DNA targeting for use in epigenomic editing and transcriptional regulation, such as specifically cleaving a target region of interest, such as a target gene, or activating or repressing gene expression of a target gene. The optimized gRNAs provide increased target binding specificity, while having decreased off-target binding and off-target activity of the CRISPR/Cas9-based and CRISPR/Cpf1-based systems by modulating lifetimes at off-target locations so as to minimize any activity at those off-target sites.

The optimized gRNA can modulate the Cas9-fusion protein activities by modulating the Cas9 lifetime at these locations and modulating the overall invasion kinetics without regard to second domain activity. In addition, gRNA binding to the protospacer at the 5′-end of the protospacer targeting segment may also be involved with Cas9 cleavage. The decreased binding to off-target sites would limit the potential for full invasion/cleavage at these off-target sites. An engineered form of the Type II effector system of Streptococcus pyogenes was shown to function in human cells for genome engineering. In this system, the Cas9 protein was directed to genomic target sites by a synthetically reconstituted “guide RNA” (“gRNA”, also used interchangeably herein as a chimeric single guide RNA (“sgRNA”)), which for Cas9 is a crRNA-tracrRNA fusion that obviates the need for RNase III and crRNA processing in general. Provided herein are CRISPR/Cas9-based systems for use in genome editing and treating genetic diseases. The CRISPR/Cas9-based systems may be designed to target any gene, including genes involved in a genetic disease, aging, tissue regeneration, or wound healing. The CRISPR/Cas9-based systems may include a Cas9 protein or Cas9 fusion protein and at least one optimized gRNA, as described below. The Cas9 fusion protein may, for example, include a domain that has a different activity that what is endogenous to Cas9, such as a transactivation domain.

The target gene may have a mutation such as a frameshift mutation or a nonsense mutation. If the target gene has a mutation that causes a premature stop codon, an aberrant splice acceptor site or an aberrant splice donor site, the CRISPR/Cas9-based system may be designed to recognize and bind a nucleotide sequence upstream or downstream from the premature stop codon, the aberrant splice acceptor site or the aberrant splice donor site. The CRISPR-Cas9-based system may also be used to disrupt normal gene splicing by targeting splice acceptors and donors to induce skipping of premature stop codons or restore a disrupted reading frame. The CRISPR/Cas9-based system may or may not mediate off-target changes to protein-coding regions of the genome.

i. Cas9

The CRISPR/Cas9-based system may include a Cas9 protein or a Cas9 fusion protein. Cas9 protein is an endonuclease that cleaves nucleic acid and is encoded by the CRISPR loci and is involved in the Type II CRISPR system. The Cas9 protein may be from any bacterial or archaea species, such as Streptococcus pyogenes. The Cas9 protein may be mutated so that the nuclease activity is inactivated. An inactivated Cas9 protein from Streptococcus pyogenes (iCas9, also referred to as “dCas9”) with no endonuclease activity has been recently targeted to genes in bacteria, yeast, and human cells by gRNAs to silence gene expression through steric hindrance. As used herein, “iCas9” and “dCas9” both refer to a Cas9 protein that has the amino acid substitutions D10A and H840A and has its nuclease activity inactivated. In some embodiments, an inactivated Cas9 protein from Neisseria meningitides, such as NmCas9, may be used. For example, the CRISPR/Cas9-based system may include a iCas9 of SEQ ID NO: 1.

ii. Cas9 Fusion Protein

The CRISPR/Cas9-based system may include a fusion protein of a Cas9 protein that does not have nuclease activity, such as dCas9, and a second domain. The second domain may include a transcription activation domain, such as a VP64 domain or p300 domain, transcription repression domain, such as KRAB domain, nuclease domain, transcription release factor domain, histone modification domain, nucleic acid association domain, acetylase domain, deacetylase domain, methylase domain, such as a DNA methylase domain, demethylase domain, phosphorylation domain, ubiquitylation domain, or sumoylation domain. The second domain may be a modifier of DNA methylation or chromatin looping.

In some embodiments, the fusion protein can include a dCas9 domain and a transcriptional activator. For example, the fusion protein can include the amino acid sequence of SEQ ID NO: 2. In other embodiments, the fusion protein can include a dCas9 domain and a transcriptional repressor. For example, the fusion protein comprises the amino acid sequence of SEQ ID NO:3. In further aspects, the fusion protein can include a dCas9 domain and a site-specific nuclease that is different from Cas9 nuclease activity.

The fusion protein may comprise two heterologous polypeptide domains, wherein the first polypeptide domain comprises a Cas protein and the second polypeptide domain has does not have nuclease activity. The fusion protein may include a Cas9 protein or a mutated Cas9 protein, as described above, fused to a second polypeptide domain that has nuclease activity. The second polypeptide domain may have nuclease activity that is different from the nuclease activity of the Cas9 protein. A nuclease, or a protein having nuclease activity, is an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acids. Nucleases are usually further divided into endonucleases and exonucleases, although some of the enzymes may fall in both categories. Well known nucleases are deoxyribonuclease and ribonuclease.

(1) CRISPR/Cas9-Based Gene Activation System

The CRISPR/Cas9-based system can be a CRISPR/Cas9-based gene activation system that can activate regulatory element function with exceptional specificity of epigenome editing. The CRISPR/Cas9-based gene activation system can be used to screen for enhancers, insulators, silencers, and locus control regions that can be targeted to increase or decrease target gene expression. This technology can be used to assign function to putative regulatory elements identified through genomic studies such as the ENCODE and the Roadmap Epigenomics projects.

The CRISPR/Cas9-based gene activation system may activate gene expression by modifying DNA methylation, chromatin looping or catalyzing acetylation of histone H3 lysine 27 at its target sites, leading to robust transcriptional activation of target genes from promoters and proximal and distal enhancers. The CRISPR/Cas9-based gene activation system is highly specific and may be guided to the target gene using as few as one guide RNA. The CRISPR/Cas9-based gene activation system may activate the expression of one gene or a family of genes by targeting enhancers at distant locations in the genome.

(a) Histone Acetyltransferase (HAT) Protein

The CRISPR/Cas9-based gene activation system may include a histone acetyltransferase protein, such as a p300 protein, CREB binding protein (CBP; an analog of p300), GCN5, or PCAF, or fragment thereof. Acetylating histones in regulatory elements using a programmable CRISPR/Cas9-based fusion protein is an effective strategy to increase the expression of target genes. A CRISPR/Cas9-based histone acetyltransferase that can be targeted to any site in the genome is uniquely capable of activating distal regulatory elements. The histone acetyltransferase protein may include a human p300 protein or a fragment thereof. The histone acetyltransferase protein may include a wild-type human p300 protein or a mutant human p300 protein, or fragments thereof. The histone acetyltransferase protein may include the core lysine-acetyltransferase domain of the human p300 protein, i.e., the p300 HAT Core (also known as “p300 Core”).

(b) CRISPR/dCas9^(p300 Core) Activation System

The p300 protein regulates the activity of many genes in tissues throughout the body. The p300 protein plays a role in regulating cell growth and division, prompting cells to mature and assume specialized functions (differentiate) and preventing the growth of cancerous tumors. The p300 protein may activate transcription by connecting transcription factors with a complex of proteins that carry out transcription in the cell's nucleus. The p300 protein also functions as a histone acetyltransferase that regulates transcription via chromatin remodeling.

The dCas9^(p300 Core) fusion protein is a potent and easily programmable tool to synthetically manipulate acetylation at targeted endogenous loci, leading to regulation of proximal and distal enhancer-regulated genes. The p300 Core acetylates lysine 27 on histone H3 (H3K27ac) and may provide H3K27ac enrichment. The fusion of the catalytic core domain of p300 to dCas9 may result in substantially higher transactivation of downstream genes than the direct fusion of full-length p300 protein despite robust protein expression. The dCas9^(p300 Core) fusion protein may also exhibit an increased transactivation capacity relative to dCas9^(VP64), including in the context of the Nm-dCas9 scaffold, especially at distal enhancer regions, at which dCas9^(VP64) displayed little, if any, measurable downstream transcriptional activity. Additionally, the dCas9^(p300 Core) displays precise and robust genome-wide transcriptional specificity. dCas9^(p300 Core) may be capable of potent transcriptional activation and co-enrichment of acetylation at promoters targeted by the epigenetically modified enhancer.

The dCas9^(p300 Core) may activate gene expression through a single gRNA that target and bind a promoter and/or a characterized enhancer. This technology also affords the ability to synthetically transactivate distal genes from putative and known regulatory regions and simplifies transactivation via the application of a single programmable effector and single target site. These capabilities allow multiplexing to target several promoters and/or enhancers simultaneously. The mammalian origin of p300 may provide advantages over virally-derived effector domains for in vivo applications by minimizing potential immunogenicity.

Gene activation by dCas9^(p300-Core) is highly specific for the target gene. In some embodiments, the p300 Core includes amino acids 1048-1664 of SEQ ID NO: 2 (i.e., SEQ ID NO: 4). In some embodiments, the CRISPR/Cas9-based gene activation system includes a dCas9^(p300 Core) fusion protein of SEQ ID NO: 2 or an Nm-dCas9^(p300 Core) fusion protein of SEQ ID NO: 5.

(2) CRISPR/Cas9-Based Gene Repression System

The CRISPR/Cas9-based system can be a CRISPR/Cas9-based gene repression system which can inhibit regulatory element function with exceptional specificity of epigenome editing. In some embodiments, the CRISPR/Cas9-based gene repression system, such as one that include dCas9^(KRAB), can interfere with distal enhancer activity by highly specific remodeling of the epigenetic state of targeted genetic loci.

(a) CRISPR/dCas9^(KRAB) Gene Repression System

The dCas9^(KRAB) repressor is a highly specific epigenome editing tool that can be used in loss-of-function screens to study gene function and discover targets for drug development. The dCas9^(KRAB) has exceptional specificity to target a particular enhancer, silence only the target genes of that enhancer, and create a repressive heterochromatin environment at that site. dCas9^(KRAB) can be used to screen for novel regulatory elements within the endogenous genomic context by silencing proximal or distal regulatory elements and corresponding gene targets. The specificity of dCas9-KRAB repressors allows it to be used for transcriptome-wide specificity for silencing endogenous genes. Epigenetic mechanisms for disruption at targeted locus such as histone methylation.

The KRAB domain, a common heterochromatin-forming motif in naturally occurring zinc finger transcription factors, has been genetically linked to dCas9 to create an RNA-guided synthetic repressor, dCas9^(KRAB). The Kruppel-associated box (“KRAB”) recruits heterochromatin-forming factors: Kap1, HP1, SETDB1, NuRD. It induces H3K0 tri-methylation, histone deacetylation. KRAB-based synthetic repressors can effectively silence the expression of single genes and have been employed to repress oncogenes, inhibit viral replication, and treat dominant negative diseases.

4. CRISPR/CPF1-BASED SYSTEM

The disclosed optimized gRNA may be used with a Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 or (“CRISPR/Cpf1”) system. CRISPR/Cpf1 system, a DNA-editing technology analogous to the CRISPR/Cas9 system, is found in Prevotella and Francisella bacteria and prevents genetic damage from viruses. Cpf1 is an RNA-guided endonuclease of a class II CRISPR/Cas system containing a 1,300 amino acid protein. Cpf1 genes are associated with the CRISPR locus, coding for an endonuclease that use a guide RNA to find and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9 and has a smaller sgRNA molecule (proximately half as many nucleotides as Cas9) as functional Cpf1 does not need the tracrRNA and only crRNA is required. Examples of Cpf1 that can be used with the optimized gRNA include Cpf1 from Acidaminococcus and Lachnospiraceae bacterial.

The Cpf1 loci encode Cas1, Cas2 and Cas4 proteins more similar to types I and III than from type II systems. The Cpf1 locus contains a mixed alpha/beta domain, a RuvC-I followed by a helical region, a RuvC-II and a zinc finger-like domain. The Cpf1 protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9. Cpf1 does not have a HNH endonuclease domain, and the N-terminal of Cpf1 does not have the alfa-helical recognition lobe of Cas9. Cpf1 CRISPR-Cas domain architecture shows that Cpf1 is functionally unique, being classified as Class 2, type V CRISPR system.

The CRISPR/Cpf1 system consists of a Cpf1 enzyme and a guide RNA that finds and positions the complex at the correct spot on the double helix to cleave target DNA. CRISPR/Cpf1 systems activity has three stages: adaptation, formation of crRNAs and interference. During the adaptation stage, Cas1 and Cas2 proteins facilitate the adaptation of small fragments of DNA into the CRISPR array. The formation of crRNAs stage involves processing of pre-cr-RNAs producing of mature crRNAs to guide the Cas protein. In the interference stage, the Cpf1 is bound to a crRNA to form a binary complex to identify and cleave a target DNA sequence.

The Cpf1-crRNA complex cleaves target DNA or RNA by identification of a protospacer adjacent motif 5′-YTN-3′ (where “Y” is a pyrimidine and “N” is any nucleobase) or 5′-TTN-3′, in contrast to the G-rich PAM targeted by Cas9. The PAM targeted by Cpf1 is on the 5′ side of the guide RNA, in contrast to the PAM targeted by Cas9, which is on the 3′ side of the guide RNA. After identification of PAM, Cpf1 introduces a sticky-end-like DNA double-stranded break of 4 or 5 nucleotides overhang in contrast to the blunt end cuts of Cas9 thereby enhancing the efficiency of genetic insertions and specificity during NHEJ or HDR. TTN PAM sites are more useful for human genomic engineering than GGN PAM sites because the human genome is more T-rich than G-rich. Protospacer-targeting segment of the gRNA for Cpf1 is at its extreme 3′-end, while Cas9 gRNAs are at its extreme 5′ end.

5. GRNA

The CRISPR/Cas9-based system or CRISPR/Cpf1-based system may include at least one gRNA, such as an optimized gRNA as described herein, which targets a nucleic acid sequence. The gRNA provides the specific targeting of the CRISPR/Cas9-based system or CRISPR/Cpf1-based system to a target region or gene. For the CRISPR/Cas9-based system, the gRNA is a fusion of two noncoding RNAs: a crRNA and a tracrRNA. The gRNA or sgRNA may target any desired DNA sequence by exchanging the sequence encoding a 20 bp protospacer which confers targeting specificity through complementary base pairing with the desired DNA target. gRNA mimics the naturally occurring crRNA:tracrRNA duplex involved in the Type II Effector system. This duplex, which may include, for example, a 42-nucleotide crRNA and a 75-nucleotide tracrRNA, acts as a guide for the Cas9 to cleave the target nucleic acid. The gRNA may target and bind a target region of a target gene. For the CRISPR/Cpf1-based system, the gRNA is a crRNA.

The CRISPR/Cas9-based system or CRISPR/Cpf1-based system may include at least one gRNA, such as an optimized gRNA described herein, wherein the gRNAs target different DNA sequences. The target DNA sequences may be overlapping. The target sequence or protospacer is followed by a PAM sequence at the 3′ end of the protospacer. Different Type II systems have differing PAM requirements. For example, the Streptococcus pyogenes Type II system uses an “NGG” sequence, where “N” can be any nucleotide.

6. METHODS OF GENERATING AN OPTIMIZED GUIDE RNA (GRNA)

The present disclosure is directed towards methods of generating optimized gRNAs, such as hairpin gRNAs (also referred to herein as “hpgRNA” and “hp-gRNA”). The optimized gRNA includes a nucleotide sequence of a full-length gRNA and nucleotides added to the 5′ end or the 3′ end of the full-length gRNA. In some embodiments, the full-length gRNA can be designed using a program such as SgRNA designer, CRISPR MultiTargeter, or SSFinder. The nucleotides added to the 5′ end for the CRISPR/Cas9 system or the 3′ end for the CRISPR/Cpf1 system of the full-length gRNA can form secondary structures by hybridizing or partially hybridizing to the nucleotides in the protospacer-targeting sequence of the full-length gRNA. The secondary structure modulates DNA binding or cleavage by disrupting invasion of the DNA duplex by the gRNA. The secondary structure influences the invasion kinetics of the gRNA rather than the binding energy of the gRNA with the complementary DNA strand. As described in the examples below, guide RNAs of type II CRISPR-Cas systems bind to protospacers through a Cas9-facilitated process known as ‘strand invasion,’ where the Cas9 protein itself first binds to and melts the protospacer adjacent motif (PAM) through direct interactions, followed by base-pairing of the 3′-end of the gRNA with the PAM-adjacent nucleotides (the ‘seed’ region) then proceeding nucleotide-by-nucleotide from the 3′- of the gRNA to the 5′-end base-pairing with the protospacer. A similar mechanism is used with the CRISPR/Cpf1 system.

The nucleotides added to the 5′ end or 3′ end of the full-length gRNA are not merely added to hybridize with the protospacer-targeting segment of the guide RNA (hairpins) to block access to the protospacer at thermodynamic equilibrium. As described in the examples, the equilibrium thermodynamic secondary structure properties (such as melting temperature of the gRNA secondary structure) are not at all correlated with the specificity of the guide RNA. Rather, in the case of cleavage and in subsequent computational work for Cas9 binding (as measured through ChIP-Seq in cells (see doi:10.1038/nbt.2916; doi:10.1038/nbt.2889)), there is a significant and substantial correlation between those and estimated strand invasion kinetics, and the structure, design, and function of guide RNAs which modulate strand invasion into the protospacer that are necessarily different than hairpins designed to compete thermodynamically for binding at equilibrium with on- and off-target sites. For example, secondary structure elements which are designed to be stable at equilibrium (such as an RNA which forms a hairpin-like structure containing internal rG-rU wobble pairs within the stem) may become rapidly destabilized during strand invasion (for example, as the rG-rU wobble pairs become the terminal base-pair of the stem as adjacent nucleotides invade the protospacer, incurring a significant energetic penalty on the RNA secondary structure, modulating the strand invasion and binding kinetics by an entirely separate mechanism than by merely blocking access to the protospacer at thermodynamic equilibrium. Secondary structures that are stable at equilibrium but rapidly destabilized during strand invasion, can be designed using the methods described herein in such a way that discriminate between on- and off-target sites with minimal thermodynamic energetic differences between the sites (a result of a single internal mismatch, say) that cannot be practically discriminated by cis-blocking or thermodynamic competition. Where invasion of the on-target site destabilizes the hairpin containing G-U wobble pairs and the sites are discriminated kinetically by invasion. For example, the VEGFA1 sites described in the examples below (the target site is GGGTGGGGGGAGTTTGCTCC, and the off-target site 2 is GGATGGAGGGAGTTTGCTCC; mismatches underlined) were able to make reduce off-target cleavage by 93% and 98% compared to a standard or full-length guide RNA or truncated guide RNA, respectively, using the computationally designed secondary structures which account for strand invasion.

Additionally, the nucleotides may be added to the 5′ end or 3′ end of a full-length gRNA to disrupt a ‘naturally-occurring’ secondary structure on the protospacer targeting segment of the gRNA in the ‘seed’ region to enhance the initiation of strand invasion by the guide RNA. Hence, the addition of these nucleotides which form secondary structures that alter strand invasion by hybridizing partially hybridizing nucleotides in the protospacer-targeting sequence to modulate DNA binding or cleavage represent a different class of guide RNA modification.

The optimized gRNAs are designed to minimize binding at an off-target site and to allow binding to a protospacer sequence. In some embodiments, the off-target site is a known or predicted off-target site. In some embodiments, the methods involve identifying a target region of interest, the target region of interest comprising a protospacer sequence; determining a polynucleotide sequence of a full-length gRNA that targets the target region of interest, the full-length gRNA comprising a protospacer-targeting sequence or segment; determining at least one or more off-target sites for the full-length gRNA; generating a polynucleotide sequence of a first gRNA, the first gRNA comprising the polynucleotide sequence of the full-length gRNA and a RNA segment, the RNA segment comprising a polynucleotide sequence having a length of M nucleotides that is complementary to a nucleotide segment of the protospacer-targeting sequence or segment, the RNA segment is at the 5′ end of the polynucleotide sequence of the full-length gRNA, the first gRNA optionally comprising a linker between the 5′ end of the polynucleotide sequence of the full-length gRNA and the RNA segment, the linker comprising a polynucleotide sequence having a length of N nucleotides, the first gRNA capable of invading the protospacer sequence and binding to a DNA sequence that is complementary to the protospacer sequence and forming a protospacer-duplex, and the first gRNA capable of invading an off-target site and binding to a DNA sequence that is complementary to the off-target site and forming an off-target duplex; calculating an estimate or computationally simulating the invasion kinetics and lifetime that the first gRNA remains invaded in the protospacer and off-target site duplexes, wherein the dynamics of invasion are estimated nucleotide-by-nucleotide by determining the energetic differences between further invasion of a different gRNA and re-annealing of the first gRNA to the DNA sequence that is complementary to the protospacer sequence; comparing the estimated lifetimes at the protospacer and/or off-target sites of the first gRNA with the estimated lifetimes of the full-length gRNA or a truncated gRNA (tru-gRNA) at the protospacer and/or off-target sites; randomizing 0 to N nucleotides in the linker and 0 to M nucleotides in the first gRNA and generating a second gRNA and repeating step (e) with the second gRNA; identifying an optimized gRNA based on a gRNA sequence that satisfy a design criteria; and testing the optimized gRNA in vivo to determine the specificity of binding.

In some embodiments, the methods involve identifying a target region of interest, the target region of interest comprising a protospacer sequence; determining a polynucleotide sequence of a full-length gRNA that targets the target region of interest, the full-length gRNA comprising a protospacer-targeting sequence or segment; determining at least one or more off-target sites for the full-length gRNA; generating a polynucleotide sequence of a first gRNA, the first gRNA comprising the polynucleotide sequence of the full-length gRNA and a RNA segment, the RNA segment comprising a polynucleotide sequence having a length of M nucleotides that is complementary to a nucleotide segment of the protospacer-targeting sequence or segment, the RNA segment is at the 3′ end of the polynucleotide sequence of the full-length gRNA, the first gRNA optionally comprising a linker between the 3′ end of the polynucleotide sequence of the full-length gRNA and the RNA segment, the linker comprising a polynucleotide sequence having a length of N nucleotides, the first gRNA capable of invading the protospacer sequence and binding to a DNA sequence that is complementary to the protospacer sequence and forming a protospacer-duplex, and the first gRNA capable of invading an off-target site and binding to a DNA sequence that is complementary to the off-target site and forming an off-target duplex; calculating an estimate or computationally simulating the invasion kinetics and lifetime that the first gRNA remains invaded in the protospacer and off-target site duplexes, wherein the dynamics of invasion are estimated nucleotide-by-nucleotide by determining the energetic differences between further invasion of a different gRNA and re-annealing of the first gRNA to the DNA sequence that is complementary to the protospacer sequence; comparing the estimated lifetimes at the protospacer and/or off-target sites of the first gRNA with the estimated lifetimes of the full-length gRNA or a truncated gRNA (tru-gRNA) at the protospacer and/or off-target sites; randomizing 0 to N nucleotides in the linker and 0 to M nucleotides in the first gRNA and generating a second gRNA and repeating step (e) with the second gRNA; identifying an optimized gRNA based on a gRNA sequence that satisfy a design criteria; and testing the optimized gRNA in vivo to determine the specificity of binding.

In some embodiments, the energetics of further invasion of a different gRNA is determined by determining the energetics of at least one of (I) breaking a DNA-DNA base-pairing, (II) forming an RNA-DNA base-pair, (III) energetic difference resulting from disrupting or forming different secondary structure within the uninvaded guide RNA, and (IV) forming or disrupting interactions between the displaced DNA strand that is complementary to the protospacer and any unpaired guide RNA nucleotides which are not involved in secondary structures. In some embodiments, the energetics of re-annealing of the first gRNA to the DNA sequence that is complementary to the protospacer sequence is determined by determining the energetics of at least one of (I) forming a DNA-DNA base-pairing, (II) breaking an RNA-DNA base-pair, (III) energetic difference resulting from disrupting or forming different secondary structure within the newly uninvaded guide RNA, and (IV) forming or disrupting interactions between the displaced DNA strand that is complementary to the protospacer and any unpaired guide RNA nucleotides which are not involved in secondary structures. In some embodiments, the method further comprises determining the energetic considerations from at least one of (V) base-pairing across mismatches, (VI) interactions with the Cas9 protein, and/or (VII) additional heuristics, wherein the additional heuristics relate to binding lifetime, extent of invasion, stability of invading guide RNA, or other calculated/simulated properties of gRNA invasion to Cas9 cleavage activity.

The CRISPR/Cas9-based system or CRISPR/Cpf1-based system can use gRNA, such as an optimized gRNA described herein, of varying sequences and lengths. In some embodiments, a full-length gRNA may comprise a protospacer-targeting segment which corresponds to the polynucleotide sequence of the target DNA sequence (i.e., protospacer). In some embodiments, the protospacer-targeting segment may have at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 30 nucleotides, or at least 35 nucleotides. The gRNA may target at least one of a promoter region, an enhancer region, a repressor region, an insulator region, a silencer region, a region involved in DNA looping with the promoter region, a gene splicing region, or the transcribed region of the target gene. In some embodiments, the full-length gRNA comprises a protospacer-targeting segment having between about 15 and 20 nucleotides.

In some embodiments, the RNA segment comprises between 2 and 20 nucleotides, between 3 and 10 nucleotides, or between 5 and 8 nucleotides. In some embodiments, the RNA segment comprises between 2 and 20 nucleotides, between 3 and 10 nucleotides, or between 5 and 8 nucleotides that complement the protospacer-targeting sequence. In some embodiments, M is between 1 and 20, between 1 and 19, between 1 and 18, between 1 and 17, between 1 and 16, between 1 and 15, between 1 and 14, between 1 and 13, between 1 and 12, between 1 and 11, between 1 and 10, between 1 and 9, between 1 and 8, between 1 and 7, between 1 and 6, between 1 and 5, between 2 and 20, between 2 and 19, between 2 and 18, between 2 and 17, between 2 and 16, between 2 and 15, between 2 and 14, between 2 and 13, between 2 and 12, between 2 and 11, between 2 and 10, between 2 and 9, between 2 and 8, between 2 and 7, between 2 and 6, between 2 and 5, between 3 and 20, between 3 and 19, between 3 and 18, between 3 and 17, between 3 and 16, between 3 and 15, between 3 and 14, between 3 and 13, between 3 and 12, between 3 and 11, between 3 and 10, between 3 and 9, between 3 and 8, between 3 and 7, between 3 and 6, between 3 and 5, between 4 and 20, between 4 and 19, between 4 and 18, between 4 and 17, between 4 and 16, between 4 and 15, between 4 and 14, between 4 and 13, between 4 and 12, between 4 and 11, between 4 and 10, between 4 and 9, between 4 and 8, between 4 and 7, between 4 and 6, between 4 and 5, between 5 and 20, between 5 and 19, between 5 and 18, between 5 and 17, between 5 and 16, between 5 and 15, between 5 and 14, between 5 and 13, between 5 and 12, between 5 and 11, between 5 and 10, between 5 and 9, between 5 and 8, between 5 and 7, between 5 and 6, between 6 and 20, between 6 and 19, between 6 and 18, between 6 and 17, between 6 and 16, between 6 and 15, between 6 and 14, between 6 and 13, between 6 and 12, between 6 and 11, between 6 and 10, between 6 and 9, between 6 and 8, between 6 and 7, between 7 and 20, between 7 and 19, between 7 and 18, between 7 and 17, between 7 and 16, between 7 and 15, between 7 and 14, between 7 and 13, between 7 and 12, between 7 and 11, between 7 and 10, between 7 and 9, between 7 and 8, between 8 and 20, between 8 and 19, between 8 and 18, between 8 and 17, between 8 and 16, between 8 and 15, between 8 and 14, between 8 and 13, between 8 and 12, between 8 and 11, between 8 and 10, between 8 and 9, between 9 and 20, between 9 and 19, between 9 and 18, between 9 and 17, between 9 and 16, between 9 and 15, between 9 and 14, between 9 and 13, between 9 and 12, between 9 and 11, or between 9 and 10. For example, M can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the RNA segment can have between 1 and 20, between 1 and 19, between 1 and 18, between 1 and 17, between 1 and 16, between 1 and 15, between 1 and 14, between 1 and 13, between 1 and 12, between 1 and 11, between 1 and 10, between 1 and 9, between 1 and 8, between 1 and 7, between 1 and 6, between 1 and 5, between 2 and 20, between 2 and 19, between 2 and 18, between 2 and 17, between 2 and 16, between 2 and 15, between 2 and 14, between 2 and 13, between 2 and 12, between 2 and 11, between 2 and 10, between 2 and 9, between 2 and 8, between 2 and 7, between 2 and 6, between 2 and 5, between 3 and 20, between 3 and 19, between 3 and 18, between 3 and 17, between 3 and 16, between 3 and 15, between 3 and 14, between 3 and 13, between 3 and 12, between 3 and 11, between 3 and 10, between 3 and 9, between 3 and 8, between 3 and 7, between 3 and 6, between 3 and 5, between 4 and 20, between 4 and 19, between 4 and 18, between 4 and 17, between 4 and 16, between 4 and 15, between 4 and 14, between 4 and 13, between 4 and 12, between 4 and 11, between 4 and 10, between 4 and 9, between 4 and 8, between 4 and 7, between 4 and 6, between 4 and 5, between 5 and 20, between 5 and 19, between 5 and 18, between 5 and 17, between 5 and 16, between 5 and 15, between 5 and 14, between 5 and 13, between 5 and 12, between 5 and 11, between 5 and 10, between 5 and 9, between 5 and 8, between 5 and 7, between 5 and 6, between 6 and 20, between 6 and 19, between 6 and 18, between 6 and 17, between 6 and 16, between 6 and 15, between 6 and 14, between 6 and 13, between 6 and 12, between 6 and 11, between 6 and 10, between 6 and 9, between 6 and 8, between 6 and 7, between 7 and 20, between 7 and 19, between 7 and 18, between 7 and 17, between 7 and 16, between 7 and 15, between 7 and 14, between 7 and 13, between 7 and 12, between 7 and 11, between 7 and 10, between 7 and 9, between 7 and 8, between 8 and 20, between 8 and 19, between 8 and 18, between 8 and 17, between 8 and 16, between 8 and 15, between 8 and 14, between 8 and 13, between 8 and 12, between 8 and 11, between 8 and 10, between 8 and 9, between 9 and 20, between 9 and 19, between 9 and 18, between 9 and 17, between 9 and 16, between 9 and 15, between 9 and 14, between 9 and 13, between 9 and 12, between 9 and 11, or between 9 and 10 nucleotides, some of which or all of which complement the protospacer-targeting sequence. In some embodiments, the RNA segment can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides.

In some embodiments, N is between 1 and 20, between 1 and 19, between 1 and 18, between 1 and 17, between 1 and 16, between 1 and 15, between 1 and 14, between 1 and 13, between 1 and 12, between 1 and 11, between 1 and 10, between 1 and 9, between 1 and 8, between 1 and 7, between 1 and 6, between 1 and 5, between 2 and 20, between 2 and 19, between 2 and 18, between 2 and 17, between 2 and 16, between 2 and 15, between 2 and 14, between 2 and 13, between 2 and 12, between 2 and 11, between 2 and 10, between 2 and 9, between 2 and 8, between 2 and 7, between 2 and 6, between 2 and 5, between 3 and 20, between 3 and 19, between 3 and 18, between 3 and 17, between 3 and 16, between 3 and 15, between 3 and 14, between 3 and 13, between 3 and 12, between 3 and 11, between 3 and 10, between 3 and 9, between 3 and 8, between 3 and 7, between 3 and 6, between 3 and 5, between 4 and 20, between 4 and 19, between 4 and 18, between 4 and 17, between 4 and 16, between 4 and 15, between 4 and 14, between 4 and 13, between 4 and 12, between 4 and 11, between 4 and 10, between 4 and 9, between 4 and 8, between 4 and 7, between 4 and 6, between 4 and 5, between 5 and 20, between 5 and 19, between 5 and 18, between 5 and 17, between 5 and 16, between 5 and 15, between 5 and 14, between 5 and 13, between 5 and 12, between 5 and 11, between 5 and 10, between 5 and 9, between 5 and 8, between 5 and 7, between 5 and 6, between 6 and 20, between 6 and 19, between 6 and 18, between 6 and 17, between 6 and 16, between 6 and 15, between 6 and 14, between 6 and 13, between 6 and 12, between 6 and 11, between 6 and 10, between 6 and 9, between 6 and 8, between 6 and 7, between 7 and 20, between 7 and 19, between 7 and 18, between 7 and 17, between 7 and 16, between 7 and 15, between 7 and 14, between 7 and 13, between 7 and 12, between 7 and 11, between 7 and 10, between 7 and 9, between 7 and 8, between 8 and 20, between 8 and 19, between 8 and 18, between 8 and 17, between 8 and 16, between 8 and 15, between 8 and 14, between 8 and 13, between 8 and 12, between 8 and 11, between 8 and 10, between 8 and 9, between 9 and 20, between 9 and 19, between 9 and 18, between 9 and 17, between 9 and 16, between 9 and 15, between 9 and 14, between 9 and 13, between 9 and 12, between 9 and 11, or between 9 and 10. For example, N can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the linker comprises between 1 and 20 nucleotides, between 3 and 10 nucleotides, or between 5 and 8 nucleotides. For example, the linker can have between 1 and 20, between 1 and 19, between 1 and 18, between 1 and 17, between 1 and 16, between 1 and 15, between 1 and 14, between 1 and 13, between 1 and 12, between 1 and 11, between 1 and 10, between 1 and 9, between 1 and 8, between 1 and 7, between 1 and 6, between 1 and 5, between 2 and 20, between 2 and 19, between 2 and 18, between 2 and 17, between 2 and 16, between 2 and 15, between 2 and 14, between 2 and 13, between 2 and 12, between 2 and 11, between 2 and 10, between 2 and 9, between 2 and 8, between 2 and 7, between 2 and 6, between 2 and 5, between 3 and 20, between 3 and 19, between 3 and 18, between 3 and 17, between 3 and 16, between 3 and 15, between 3 and 14, between 3 and 13, between 3 and 12, between 3 and 11, between 3 and 10, between 3 and 9, between 3 and 8, between 3 and 7, between 3 and 6, between 3 and 5, between 4 and 20, between 4 and 19, between 4 and 18, between 4 and 17, between 4 and 16, between 4 and 15, between 4 and 14, between 4 and 13, between 4 and 12, between 4 and 11, between 4 and 10, between 4 and 9, between 4 and 8, between 4 and 7, between 4 and 6, between 4 and 5, between 5 and 20, between 5 and 19, between 5 and 18, between 5 and 17, between 5 and 16, between 5 and 15, between 5 and 14, between 5 and 13, between 5 and 12, between 5 and 11, between 5 and 10, between 5 and 9, between 5 and 8, between 5 and 7, between 5 and 6, between 6 and 20, between 6 and 19, between 6 and 18, between 6 and 17, between 6 and 16, between 6 and 15, between 6 and 14, between 6 and 13, between 6 and 12, between 6 and 11, between 6 and 10, between 6 and 9, between 6 and 8, between 6 and 7, between 7 and 20, between 7 and 19, between 7 and 18, between 7 and 17, between 7 and 16, between 7 and 15, between 7 and 14, between 7 and 13, between 7 and 12, between 7 and 11, between 7 and 10, between 7 and 9, between 7 and 8, between 8 and 20, between 8 and 19, between 8 and 18, between 8 and 17, between 8 and 16, between 8 and 15, between 8 and 14, between 8 and 13, between 8 and 12, between 8 and 11, between 8 and 10, between 8 and 9, between 9 and 20, between 9 and 19, between 9 and 18, between 9 and 17, between 9 and 16, between 9 and 15, between 9 and 14, between 9 and 13, between 9 and 12, between 9 and 11, or between 9 and 10 nucleotides, some of which or all of which complement the protospacer-targeting sequence. In some embodiments, the linker can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, the linker can include a stabilizing linker, such as a tetraloop. Examples of tetraloop, include but are not limited to ANYA, CUYG, GNRA, UMAC and UNCG.

In some embodiments, the RNA segment and/or protospacer-targeting sequence provide a secondary structure. In some embodiments, the secondary structure is formed by partially hybridizing the protospacer-targeting sequence with the RNA segment. In some embodiments, the secondary structure modulates DNA binding or cleavage by Cas9 by disrupting invasion of the protospacer duplex or off-target duplex by the optimized gRNA. In some embodiments, the secondary structure keeps the 5′-end of the gRNA stably within the protein and protects the optimized gRNA within the Cas9 to prevent degradation

In some embodiments, the secondary structure is formed by hybridizing all or part of the RNA segment to nucleotides in the 5′ end of the protospacer-targeting sequence or segment, nucleotides in the middle of the protospacer-targeting sequence or segment, and/or nucleotides in the 3′-end of the protospacer-targeting sequence or segment. In some embodiments, contiguous segments of the RNA segment hybridize to the protospacer-targeting sequence or segment. In some embodiments, non-contiguous segment of the RNA segment hybridize to the protospacer-targeting sequence or segment. In some embodiments, the secondary structure is a hairpin.

In some embodiments, the secondary structure is stable at room temperature or 37° C. In some embodiments, overall equilibrium free energy of the secondary structure is less than about 2 kcal/mol at a temperature between about 4° C. and about 50° C., such as room temperature or 37° C. For example, the overall equilibrium free energy of the secondary structure can be less than about 10 kcal/mol, less than about 5 kcal/mol, less than about 4 kcal/mol, less than about 3 kcal/mol, less than about 2 kcal/mol, less than about 1 kcal/mol, or less than about 0.5 kcal/mol at a temperature between about 4° C. and about 50° C., between about 4° C. and about 40° C., between about 4° C. and about 37° C., between about 4° C. and about 30° C., between about 4° C. and about 25° C., between about 4° C. and about 20° C., between about 4° C. and about 10° C., between about 5° C. and about 50° C., between about 5° C. and about 40° C., between about 5° C. and about 37° C., between about 5° C. and about 30° C., between about 5° C. and about 25° C., between about 5° C. and about 20° C., between about 5° C. and about 10° C., between about 10° C. and about 50° C., between about 10° C. and about 40° C., between about 10° C. and about 37° C., between about 10° C. and about 30° C., between about 10° C. and about 25° C., between about 10° C. and about 20° C., between about 20° C. and about 50° C., between about 20° C. and about 40° C., between about 20° C. and about 37° C., between about 20° C. and about 30° C., between about 25° C. and about 50° C., between about 25° C. and about 40° C., between about 25° C. and about 37° C., or between about 25° C. and about 30° C. In some embodiments, the RNA segment hybridizes or forms non-canonical base pairs with at least two nucleotides of the protospacer-targeting sequence or segment. In some embodiments, the non-canonical base pair is rU-rG.

In some embodiments, between 1 and 20 nucleotides are randomized in the linker. For example, between 1 and 20, between 1 and 15, between 1 and 10, between 1 and 9, between 1 and 8, between 1 and 7, between 1 and 6, between 1 and 5, between 1 and 4, between 1 and 3, between 1 and 2, between 2 and 20, between 2 and 15, between 2 and 10, between 2 and 9, between 2 and 8, between 2 and 7, between 2 and 6, between 2 and 5, between 2 and 4, between 3 and 20, between 3 and 15, between 3 and 10, between 3 and 9, between 3 and 8, between 3 and 7, between 3 and 6, between 3 and 5, between 3 and 4, between 4 and 20, between 4 and 15, between 4 and 10, between 4 and 9, between 4 and 8, between 4 and 7, between 4 and 6, between 4 and 5, between 5 and 20, between 5 and 15, between 5 and 10, between 5 and 9, between 5 and 8, between 5 and 7, between 5 and 6, between 6 and 20, between 6 and 15, between 6 and 10, between 6 and 9, between 6 and 8, between 6 and 7, between 7 and 20, between 7 and 15, between 7 and 10, between 7 and 9, between 7 and 8, between 8 and 20, between 8 and 15, between 8 and 10, between 8 and 9, between 9 and 20, between 9 and 15, or between 9 and 10, between 10 and 20, between 10 and 15, or between 15 and 20 nucleotides may be randomized in the linker.

In some embodiments, the between 1 and 20 nucleotides are randomized in the RNA segment. For example, between 1 and 20, between 1 and 15, between 1 and 10, between 1 and 9, between 1 and 8, between 1 and 7, between 1 and 6, between 1 and 5, between 1 and 4, between 1 and 3, between 1 and 2, between 2 and 20, between 2 and 15, between 2 and 10, between 2 and 9, between 2 and 8, between 2 and 7, between 2 and 6, between 2 and 5, between 2 and 4, between 3 and 20, between 3 and 15, between 3 and 10, between 3 and 9, between 3 and 8, between 3 and 7, between 3 and 6, between 3 and 5, between 3 and 4, between 4 and 20, between 4 and 15, between 4 and 10, between 4 and 9, between 4 and 8, between 4 and 7, between 4 and 6, between 4 and 5, between 5 and 20, between 5 and 15, between 5 and 10, between 5 and 9, between 5 and 8, between 5 and 7, between 5 and 6, between 6 and 20, between 6 and 15, between 6 and 10, between 6 and 9, between 6 and 8, between 6 and 7, between 7 and 20, between 7 and 15, between 7 and 10, between 7 and 9, between 7 and 8, between 8 and 20, between 8 and 15, between 8 and 10, between 8 and 9, between 9 and 20, between 9 and 15, or between 9 and 10, between 10 and 20, between 10 and 15, or between 15 and 20 nucleotides may be randomized in the RNA segment.

In some embodiments, step (g) is repeated X number of times, thereby generating X number of gRNAs and repeating step (e) with each X number of gRNAs, wherein X is between 0 to 20. In some embodiments, X can be is between 1 and 20, between 1 and 19, between 1 and 18, between 1 and 17, between 1 and 16, between 1 and 15, between 1 and 14, between 1 and 13, between 1 and 12, between 1 and 11, between 1 and 10, between 1 and 9, between 1 and 8, between 1 and 7, between 1 and 6, between 1 and 5, between 2 and 20, between 2 and 19, between 2 and 18, between 2 and 17, between 2 and 16, between 2 and 15, between 2 and 14, between 2 and 13, between 2 and 12, between 2 and 11, between 2 and 10, between 2 and 9, between 2 and 8, between 2 and 7, between 2 and 6, between 2 and 5, between 3 and 20, between 3 and 19, between 3 and 18, between 3 and 17, between 3 and 16, between 3 and 15, between 3 and 14, between 3 and 13, between 3 and 12, between 3 and 11, between 3 and 10, between 3 and 9, between 3 and 8, between 3 and 7, between 3 and 6, between 3 and 5, between 4 and 20, between 4 and 19, between 4 and 18, between 4 and 17, between 4 and 16, between 4 and 15, between 4 and 14, between 4 and 13, between 4 and 12, between 4 and 11, between 4 and 10, between 4 and 9, between 4 and 8, between 4 and 7, between 4 and 6, between 4 and 5, between 5 and 20, between 5 and 19, between 5 and 18, between 5 and 17, between 5 and 16, between 5 and 15, between 5 and 14, between 5 and 13, between 5 and 12, between 5 and 11, between 5 and 10, between 5 and 9, between 5 and 8, between 5 and 7, between 5 and 6, between 6 and 20, between 6 and 19, between 6 and 18, between 6 and 17, between 6 and 16, between 6 and 15, between 6 and 14, between 6 and 13, between 6 and 12, between 6 and 11, between 6 and 10, between 6 and 9, between 6 and 8, between 6 and 7, between 7 and 20, between 7 and 19, between 7 and 18, between 7 and 17, between 7 and 16, between 7 and 15, between 7 and 14, between 7 and 13, between 7 and 12, between 7 and 11, between 7 and 10, between 7 and 9, between 7 and 8, between 8 and 20, between 8 and 19, between 8 and 18, between 8 and 17, between 8 and 16, between 8 and 15, between 8 and 14, between 8 and 13, between 8 and 12, between 8 and 11, between 8 and 10, between 8 and 9, between 9 and 20, between 9 and 19, between 9 and 18, between 9 and 17, between 9 and 16, between 9 and 15, between 9 and 14, between 9 and 13, between 9 and 12, between 9 and 11, or between 9 and 10. For example, X can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

In some embodiments, the invasion kinetics and lifetime are calculated using kinetic Monte Carlo method or Gillespie algorithm. In some embodiment, the invasion kinetics and lifetime can be determined using ‘deterministic’ methods such as differential equations which model strand invasion, which are known to one of skill in the art. The kinetic Monte Carlo (KMC) method is a Monte Carlo method computer simulation intended to simulate the time evolution of some processes occurring in nature. The processes are typically processes that occur with known transition rates among states. These known transition rates are inputs to the KMC algorithm. The Gillespie algorithm (also known as the Doob-Gillespie algorithm) generates a statistically correct trajectory (possible solution) of a stochastic equation. The Gillespie algorithm can be used to simulate increasingly complex systems. The algorithm is particularly useful for simulating reactions within cells where the number of reagents typically number in the tens of molecules (or less). Mathematically, it is a variety of a dynamic Monte Carlo method and similar to the kinetic Monte Carlo methods. The Gillespie algorithm allows a discrete and stochastic simulation of a system with few reactants because every reaction is explicitly simulated. A trajectory corresponding to a single Gillespie simulation represents an exact sample from the probability mass function that is the solution of the master equation.

In some embodiments, the design criteria can be specificity, modulation of binding lifetime, and/or estimated cleavage specificity. For example, the optimized gRNA may be designed to have a binding lifetime greater than or equal to that of the full gRNA at an on-target site, and/or a binding lifetime less than or equal to that of the full-length gRNA at an off-target site. In some embodiments, the optimized gRNA is selected to have a binding lifetime less than or equal to that of the full-length gRNA to at least three off-target sites, wherein the off-target sites are predicted to be the closest off-target sites or predicted to have the highest identity to the on-target sites. In some embodiments, the design criteria comprises a lifetime or cleavage rate at an off-target site that is less than or equal to the lifetime or cleavage rate of a full-length gRNA or truncated gRNA at the off-target site and/or a predicted on-target activity rate that is greater than 10% of the predicted on-target activity rate of a full-length gRNA or truncated gRNA.

In some embodiments, the optimized gRNA is tested in step i) using a mismatch-sensitive nuclease to determine CRISPR activity, such as using surveyor assay or T7 endonuclease I (T7E1) assay, or next-gen sequencing techniques, such as Illumina MiSeq or GUIDE-Seq. In some embodiments, the optimized gRNA is tested in step i) using a reporter assay, wherein the Cas9-fusion protein activity alters the expression of a reporter protein, such as GFP. GUIDE-Seq is an assay that has been devised to assay off-target cleavages.

In some embodiments, the target region can be determined based on a sequence's proximity to a PAM sequence using a program, such as CRISPR design (Ran, et al. Nature Protocols (2013) 8:2281-2308) and CCTop (Stemmer, PLoS One (2015) 10:e0124633) tools. In some embodiments, the target sites can include promoters, DNAse I hypersensitivity sites, Transposase-Accessible Chromatin sites, DNA methylation sites, transcription factor binding sites, epigenetic marks, expression quantitative trait loci, and/or regions associated with human traits or phenotypes in genetic association studies. The target sites can be determined by DNase-sequencing (DNase-seq), Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-seq), ChIP-sequencing, self-transcribing active regulatory region sequencing (STARR-Seq), single molecule real time sequencing (SMRT), Formaldehyde-Assisted Isolation of Regulatory Elements sequencing (FAIRE-seq), micrococcal nuclease sequencing (MNase-seq), reduced representation bisulfite sequencing (RRBS-seq), whole genome bisulfite sequencing, methyl-binding DNA immunoprecipitation (MEDIP-seq), or genetic association studies. In some embodiments, the off-target site can be determined using CasOT (PKU Zebrafish Functional Genomics group, Peking University), CHOPCHOP (Harvard University), CRISPR Design, (Massachusetts Institute of Technology), CRISPR Design tool (The Broad Institute of Harvard and MIT), CRISPR/Cas9 gRNA finder (University of Colorado), CRISPRfinder (Universite Paris-Sud), E-CRISP (DKFZ German Cancer Research Center), CRISPR gRNA Design tool (DNA 2.0), PROGNOS (Emory University/Georgia Institute of Technology), ZiFiT (Massachusetts General Hospital). Examples of tools that can be used to determine target regions and off-target sites are described in International Patent Application No. WO2016109255, which is incorporated herein by reference in its entirety.

7. TARGET GENE

As disclosed herein, the CRISPR/Cas9-based system or CRISPR/Cpf1-based system may be designed to target and cleave any target gene. For example, the gRNA, such as the optimized gRNA described herein, may target and bind a target region in a target gene. The target gene may be an endogenous gene, a transgene, or a viral gene in a cell line. In some embodiments, the target gene may be a known gene. In some embodiments, the target gene is an unknown gene. The gRNA may target any nucleic acid sequence. The nucleic acid sequence target may be DNA. The DNA may be any gene. For example, the gRNA may target a gene, such as DMD, EMX1, or VEGFA.

In some aspects, the target gene is a disease-relevant gene. In some embodiments, the target cell is a mammalian cell. In some embodiments, the genome includes a human genome. In some embodiments, the target gene may be a prokaryotic gene or a eukaryotic gene, such as a mammalian gene. For example, the CRISPR/Cas9-based system or CRISPR/Cpf1-based system may target a mammalian gene, such as DMD (dystrophin gene), EMX1, VEGFA, IL1RN, MYOD1, OCT4, HBE, HBG, HBD, HBB, MYOCD (Myocardin), PAX7 (Paired box protein Pax-7), FGF1 (fibroblast growth factor-1) genes, such as FGF1A, FGF1B, and FGF1C. Other target genes include, but not limited to, Atf3, Axud1, Btg2, c-Fos, c-Jun, Cxcl1, Cxcl2, Edn1, Ereg, Fos, Gadd45b, Ier2, Ier3, Ifrd1, Il1b, 116, Irf1, Junb, Lif, Nfkbia, Nfkbiz, Ptgs2, Slc25a25, Sqstm1, Tieg, Tnf, Tnfaip3, Zfp36, Birc2, Ccl2, Ccl20, Ccl7, Cebpd, Ch25h, CSF1, Cx3cl1, Cxcl10, Cxcl5, Gch, Icam1, Ifi47, Ifngr2, Mmp10, Nfkbie, Npal1, p21, Relb, Ripk2, Rnd1, S1pr3, Stx11, Tgtp, Tlr2, Tmem140, Tnfaip2, Tnfrsf6, Vcam1, 1110004C05Rik (GenBank accession number BC010291), Abca1, AI561871 (GenBank accession number BI143915), AI882074 (GenBank accession number BB730912), Arts1, AW049765 (GenBank accession number BC026642.1), C3, Casp4, Cc15, Cc19, Cdsn, Enpp2, Gbp2, H2-D1, H2-K, H2-L, Ifit1, Ii, Il13ra1, Il1rl1, Lcn2, Lhfpl2, LOC677168 (GenBank accession number AK019325), Mmp13, Mmp3, Mt2, Naf1, Ppicap, Prnd, Psmb10, Saa3, Serpina3g, Serpinf1, Sod3, Stat1, Tapbp, U90926 (GenBank accession number NM_020562), Ubd, A2AR (Adenosine A2A receptor), B7-H3 (also called CD276), B7-H4 (also called VTCN1), BTLA (B and T Lymphocyte Attenuator; also called CD272), CTLA-4 (Cytotoxic T-Lymphocyte-Associated protein 4; also called CD152), IDO (Indoleamine 2,3-dioxygenase) KIR (Killer-cell Immunoglobulin-like Receptor), LAG3 (Lymphocyte Activation Gene-3), PD-1 (Programmed Death 1 (PD-1) receptor), TIM-3 (T-cell Immunoglobulin domain and Mucin domain 3), and VISTA (V-domain Ig suppressor of T cell activation. In some embodiments, the target gene is DMD (dystrophin), EMXJ, or VEGFA gene.

8. COMPOSITIONS FOR GENOME EDITING

The present invention is directed to compositions for genome editing, genomic alteration or altering gene expression of a target gene. The compositions include an optimized gRNA generated by the disclosed method with a a CRISPR/Cas9-based system or CRISPR/Cpf1-based system. In some embodiments, the gRNA can discriminate between on- and off-target sites with minimal thermodynamic energetic differences between the sites and provide increased specificity. In some embodiments, the optimized gRNA modulates strand invasion into the protospacer.

The increase in specificity is achieved by adding an extension to the 5′-end or 3′-end of a full-length or standard gRNA such that it forms a ‘hairpin’ structure that is self-complementary to the segment of the full-length or standard gRNA which targets the protospacer, e.g., the protospacer-targeting sequence. See FIG. 1B and FIG. 2B. The hairpins serve as a kinetic barrier to strand invasion of the protospacer, but the hairpins are displaced during strand invasion of the full target sites so full invasion can occur.

As shown in FIG. 2D, binding by dCas9 to full protospacers preferentially occurs, strongly suggesting that the hairpins are in fact displaced during invasion. The disclosed optimized gRNAs that are hairpins were designed to increase specificity in binding to targeted sites by inhibiting invasion if there were mismatches between the target and the PAM-distal targeting region of the guide RNA. In those cases, it is more energetically favorable for the hairpins to remain closed, and the presence of the hairpin likely promotes melting and detachment of Cas9/dCas9 from those sites.

Optimized gRNAs with 5′-hairpins or 3′-hairpins (hpgRNAs) significantly enhanced specificity in binding compared to both standard guide RNAs and the best available guide RNA variants (see examples), and abolished or significantly weakened binding at protospacer sites containing mismatches. Increasing lengths of the hairpin increased the specificity of dCas9 binding. Optimized gRNA and hpgRNAs can be used to tune Cas9/dCas9 or Cpf1 binding affinities and specificity. Based on the size and structure of the hairpin, the hairpin of hpgRNAs could be accommodated within the DNA-binding channel of Cas9/dCas9 molecule and protected from degradation. In some embodiments, the hairpin length, loop length, and loop composition may be changed to allow for more fine control of these properties. In some embodiments, the hairpin length can be between about 1 and about 20 nucleotides or between about 3 to about 10 nucleotides. For example, the hairpin length can be between 1 and 20, between 1 and 19, between 1 and 18, between 1 and 17, between 1 and 16, between 1 and 15, between 1 and 14, between 1 and 13, between 1 and 12, between 1 and 11, between 1 and 10, between 1 and 9, between 1 and 8, between 1 and 7, between 1 and 6, between 1 and 5, between 2 and 20, between 2 and 19, between 2 and 18, between 2 and 17, between 2 and 16, between 2 and 15, between 2 and 14, between 2 and 13, between 2 and 12, between 2 and 11, between 2 and 10, between 2 and 9, between 2 and 8, between 2 and 7, between 2 and 6, between 2 and 5, between 3 and 20, between 3 and 19, between 3 and 18, between 3 and 17, between 3 and 16, between 3 and 15, between 3 and 14, between 3 and 13, between 3 and 12, between 3 and 11, between 3 and 10, between 3 and 9, between 3 and 8, between 3 and 7, between 3 and 6, between 3 and 5, between 4 and 20, between 4 and 19, between 4 and 18, between 4 and 17, between 4 and 16, between 4 and 15, between 4 and 14, between 4 and 13, between 4 and 12, between 4 and 11, between 4 and 10, between 4 and 9, between 4 and 8, between 4 and 7, between 4 and 6, between 4 and 5, between 5 and 20, between 5 and 19, between 5 and 18, between 5 and 17, between 5 and 16, between 5 and 15, between 5 and 14, between 5 and 13, between 5 and 12, between 5 and 11, between 5 and 10, between 5 and 9, between 5 and 8, between 5 and 7, between 5 and 6, between 6 and 20, between 6 and 19, between 6 and 18, between 6 and 17, between 6 and 16, between 6 and 15, between 6 and 14, between 6 and 13, between 6 and 12, between 6 and 11, between 6 and 10, between 6 and 9, between 6 and 8, between 6 and 7, between 7 and 20, between 7 and 19, between 7 and 18, between 7 and 17, between 7 and 16, between 7 and 15, between 7 and 14, between 7 and 13, between 7 and 12, between 7 and 11, between 7 and 10, between 7 and 9, between 7 and 8, between 8 and 20, between 8 and 19, between 8 and 18, between 8 and 17, between 8 and 16, between 8 and 15, between 8 and 14, between 8 and 13, between 8 and 12, between 8 and 11, between 8 and 10, between 8 and 9, between 9 and 20, between 9 and 19, between 9 and 18, between 9 and 17, between 9 and 16, between 9 and 15, between 9 and 14, between 9 and 13, between 9 and 12, between 9 and 11, or between 9 and 10. For example, the hairpin length can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or about 5 to about 8 nucleotides.

In some embodiments, the loop length can be between about 1 and about 20 nucleotides, between about 3 to about 10 nucleotides, or between about 5 to about 8 nucleotides. For example, the loop length can be between 1 and 20, between 1 and 19, between 1 and 18, between 1 and 17, between 1 and 16, between 1 and 15, between 1 and 14, between 1 and 13, between 1 and 12, between 1 and 11, between 1 and 10, between 1 and 9, between 1 and 8, between 1 and 7, between 1 and 6, between 1 and 5, between 2 and 20, between 2 and 19, between 2 and 18, between 2 and 17, between 2 and 16, between 2 and 15, between 2 and 14, between 2 and 13, between 2 and 12, between 2 and 11, between 2 and 10, between 2 and 9, between 2 and 8, between 2 and 7, between 2 and 6, between 2 and 5, between 3 and 20, between 3 and 19, between 3 and 18, between 3 and 17, between 3 and 16, between 3 and 15, between 3 and 14, between 3 and 13, between 3 and 12, between 3 and 11, between 3 and 10, between 3 and 9, between 3 and 8, between 3 and 7, between 3 and 6, between 3 and 5, between 4 and 20, between 4 and 19, between 4 and 18, between 4 and 17, between 4 and 16, between 4 and 15, between 4 and 14, between 4 and 13, between 4 and 12, between 4 and 11, between 4 and 10, between 4 and 9, between 4 and 8, between 4 and 7, between 4 and 6, between 4 and 5, between 5 and 20, between 5 and 19, between 5 and 18, between 5 and 17, between 5 and 16, between 5 and 15, between 5 and 14, between 5 and 13, between 5 and 12, between 5 and 11, between 5 and 10, between 5 and 9, between 5 and 8, between 5 and 7, between 5 and 6, between 6 and 20, between 6 and 19, between 6 and 18, between 6 and 17, between 6 and 16, between 6 and 15, between 6 and 14, between 6 and 13, between 6 and 12, between 6 and 11, between 6 and 10, between 6 and 9, between 6 and 8, between 6 and 7, between 7 and 20, between 7 and 19, between 7 and 18, between 7 and 17, between 7 and 16, between 7 and 15, between 7 and 14, between 7 and 13, between 7 and 12, between 7 and 11, between 7 and 10, between 7 and 9, between 7 and 8, between 8 and 20, between 8 and 19, between 8 and 18, between 8 and 17, between 8 and 16, between 8 and 15, between 8 and 14, between 8 and 13, between 8 and 12, between 8 and 11, between 8 and 10, between 8 and 9, between 9 and 20, between 9 and 19, between 9 and 18, between 9 and 17, between 9 and 16, between 9 and 15, between 9 and 14, between 9 and 13, between 9 and 12, between 9 and 11, or between 9 and 10. In some embodiments, the loop length can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or about 5 to about 8 nucleotides.

In some embodiments, the loop composition can be between about 1 and about 20 nucleotides, between about 3 to about 10 nucleotides, or about 5 to about 8 nucleotides. For example, the loop composition can be between 1 and 20, between 1 and 19, between 1 and 18, between 1 and 17, between 1 and 16, between 1 and 15, between 1 and 14, between 1 and 13, between 1 and 12, between 1 and 11, between 1 and 10, between 1 and 9, between 1 and 8, between 1 and 7, between 1 and 6, between 1 and 5, between 2 and 20, between 2 and 19, between 2 and 18, between 2 and 17, between 2 and 16, between 2 and 15, between 2 and 14, between 2 and 13, between 2 and 12, between 2 and 11, between 2 and 10, between 2 and 9, between 2 and 8, between 2 and 7, between 2 and 6, between 2 and 5, between 3 and 20, between 3 and 19, between 3 and 18, between 3 and 17, between 3 and 16, between 3 and 15, between 3 and 14, between 3 and 13, between 3 and 12, between 3 and 11, between 3 and 10, between 3 and 9, between 3 and 8, between 3 and 7, between 3 and 6, between 3 and 5, between 4 and 20, between 4 and 19, between 4 and 18, between 4 and 17, between 4 and 16, between 4 and 15, between 4 and 14, between 4 and 13, between 4 and 12, between 4 and 11, between 4 and 10, between 4 and 9, between 4 and 8, between 4 and 7, between 4 and 6, between 4 and 5, between 5 and 20, between 5 and 19, between 5 and 18, between 5 and 17, between 5 and 16, between 5 and 15, between 5 and 14, between 5 and 13, between 5 and 12, between 5 and 11, between 5 and 10, between 5 and 9, between 5 and 8, between 5 and 7, between 5 and 6, between 6 and 20, between 6 and 19, between 6 and 18, between 6 and 17, between 6 and 16, between 6 and 15, between 6 and 14, between 6 and 13, between 6 and 12, between 6 and 11, between 6 and 10, between 6 and 9, between 6 and 8, between 6 and 7, between 7 and 20, between 7 and 19, between 7 and 18, between 7 and 17, between 7 and 16, between 7 and 15, between 7 and 14, between 7 and 13, between 7 and 12, between 7 and 11, between 7 and 10, between 7 and 9, between 7 and 8, between 8 and 20, between 8 and 19, between 8 and 18, between 8 and 17, between 8 and 16, between 8 and 15, between 8 and 14, between 8 and 13, between 8 and 12, between 8 and 11, between 8 and 10, between 8 and 9, between 9 and 20, between 9 and 19, between 9 and 18, between 9 and 17, between 9 and 16, between 9 and 15, between 9 and 14, between 9 and 13, between 9 and 12, between 9 and 11, or between 9 and 10. In some embodiments, the loop composition can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or about 5 to about 8 nucleotides.

The compositions may include a may include viral vector and a CRISPR/Cas9-based system or CRISPR/Cpf1-based system with at least one gRNA, such as an optimized gRNA described herein. In some embodiments, the composition includes a modified AAV vector and a nucleotide sequence encoding a CRISPR/Cas9-based system with at least one gRNA, such as an optimized gRNA described herein. The composition may further comprise a donor DNA or a transgene. These compositions may be used in genome editing, genome engineering, and correcting or reducing the effects of mutations in genes involved in genetic diseases.

The target gene may be involved in differentiation of a cell or any other process in which activation, repression, or disruption of a gene may be desired, or may have a mutation such as a deletion, frameshift mutation, or a nonsense mutation. If the target gene has a mutation that causes a premature stop codon, an aberrant splice acceptor site or an aberrant splice donor site, the CRISPR/Cas9-based system or CRISPR/Cpf1-based system with at least one gRNA, such as an optimized gRNA described herein, may be designed to recognize and bind a nucleotide sequence upstream or downstream from the premature stop codon, the aberrant splice acceptor site or the aberrant splice donor site. The CRISPR/Cas9-based system or CRISPR/Cpf1-based system with at least one gRNA, such as an optimized gRNA described herein, may also be used to disrupt normal gene splicing by targeting splice acceptors and donors to induce skipping of premature stop codons or restore a disrupted reading frame. The CRISPR/Cas9-based system or CRISPR/Cpf1-based system with at least one gRNA, such as an optimized gRNA described herein, may or may not mediate off-target changes to protein-coding regions of the genome.

In some embodiments, the CRISPR/Cas9-based system induces or represses the gene expression of a target gene by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least about 110 fold, at least 120 fold, at least 130 fold, at least 140 fold, at least 150 fold, at least 160 fold, at least 170 fold, at least 180 fold, at least 190 fold, at least 200 fold, at least about 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 fold, at least 1000 fold, at least 1500 fold, at least 2000 fold, at least 2500 fold, at least 3000 fold, at least 3500 fold, at least 4000 fold, at least 4500 fold, at least 5000 fold, at least 600 fold, at least 7000 fold, at least 8000 fold, at least 9000 fold, at least 10000 fold, at least 100000 fold compared to a control level of gene expression. A control level of gene expression of the target gene may be the level of gene expression of the target gene in a cell that is not treated with any CRISPR/Cas9-based system.

a. Modified Lentiviral Vector

The compositions for genome editing, genomic alteration or altering gene expression of a target gene may include a modified lentiviral vector. The modified lentiviral vector includes a first polynucleotide sequence encoding a DNA targeting system and a second polynucleotide sequence encoding at least one sgRNA. The first polynucleotide sequence may be operably linked to a promoter. The promoter may be a constitutive promoter, an inducible promoter, a repressible promoter, or a regulatable promoter.

The second polynucleotide sequence encodes at least 1 gRNA, such as an optimized gRNA described herein. For example, the second polynucleotide sequence may encode at least 1 gRNA, at least 2 gRNAs, at least 3 gRNAs, at least 4 gRNAs, at least 5 gRNAs, at least 6 gRNAs, at least 7 gRNAs, at least 8 gRNAs, at least 9 gRNAs, at least 10 gRNAs, at least 11 gRNA, at least 12 gRNAs, at least 13 gRNAs, at least 14 gRNAs, at least 15 gRNAs, at least 16 gRNAs, at least 17 gRNAs, at least 18 gRNAs, at least 19 gRNAs, at least 20 gRNAs, at least 25 gRNA, at least 30 gRNAs, at least 35 gRNAs, at least 40 gRNAs, at least 45 gRNAs, or at least 50 gRNAs. The second polynucleotide sequence may encode between 1 gRNA and 50 gRNAs, between 1 gRNA and 45 gRNAs, between 1 gRNA and 40 gRNAs, between 1 gRNA and 35 gRNAs, between 1 gRNA and 30 gRNAs, between 1 gRNA and 25 different gRNAs, between 1 gRNA and 20 gRNAs, between 1 gRNA and 16 gRNAs, between 1 gRNA and 8 different gRNAs, between 4 different gRNAs and 50 different gRNAs, between 4 different gRNAs and 45 different gRNAs, between 4 different gRNAs and 40 different gRNAs, between 4 different gRNAs and 35 different gRNAs, between 4 different gRNAs and 30 different gRNAs, between 4 different gRNAs and 25 different gRNAs, between 4 different gRNAs and 20 different gRNAs, between 4 different gRNAs and 16 different gRNAs, between 4 different gRNAs and 8 different gRNAs, between 8 different gRNAs and 50 different gRNAs, between 8 different gRNAs and 45 different gRNAs, between 8 different gRNAs and 40 different gRNAs, between 8 different gRNAs and 35 different gRNAs, between 8 different gRNAs and 30 different gRNAs, between 8 different gRNAs and 25 different gRNAs, between 8 different gRNAs and 20 different gRNAs, between 8 different gRNAs and 16 different gRNAs, between 16 different gRNAs and 50 different gRNAs, between 16 different gRNAs and 45 different gRNAs, between 16 different gRNAs and 40 different gRNAs, between 16 different gRNAs and 35 different gRNAs, between 16 different gRNAs and 30 different gRNAs, between 16 different gRNAs and 25 different gRNAs, or between 16 different gRNAs and 20 different gRNAs. Each of the polynucleotide sequences encoding the different gRNAs may be operably linked to a promoter. The promoters that are operably linked to the different gRNAs may be the same promoter. The promoters that are operably linked to the different gRNAs may be different promoters. The promoter may be a constitutive promoter, an inducible promoter, a repressible promoter, or a regulatable promoter. At least one gRNA may bind to a target gene or loci. If more than one gRNA is included, each of the gRNAs binds to a different target region within one target loci or each of the gRNA binds to a different target region within different gene loci.

b. Adeno-Associated Virus Vectors

AAV may be used to deliver the compositions to the cell using various construct configurations. For example, AAV may deliver a CRISPR/Cas9-based system or CRISPR/Cpf1-based system and gRNA expression cassettes on separate vectors. Alternatively, if the small Cas9 proteins, derived from species such as Staphylococcus aureus or Neisseria meningitidis, are used then both the Cas9 and up to two gRNA expression cassettes may be combined in a single AAV vector within the 4.7 kb packaging limit.

The composition, as described above, includes a modified adeno-associated virus (AAV) vector. The modified AAV vector may be capable of delivering and expressing the CRISPR/Cas9-based system or CRISPR/Cpf1-based system in the cell of a mammal. For example, the modified AAV vector may be an AAV-SASTG vector (Piacentino et al. (2012) Human Gene Therapy 23:635-646). The modified AAV vector may be based on one or more of several capsid types, including AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9. The modified AAV vector may be based on AAV2 pseudotype with alternative muscle-tropic AAV capsids, such as AAV2/1, AAV2/6, AAV2/7, AAV2/8, AAV2/9, AAV2.5 and AAV/SASTG vectors that efficiently transduce skeletal muscle or cardiac muscle by systemic and local delivery (Seto et al. Current Gene Therapy (2012) 12:139-151).

9. TARGET CELLS

As disclosed herein, the gRNA, such as an optimized gRNA described herein, may be used with a CRISPR/Cas9 system with any type of cell. In some embodiments, the cell is a bacterial cell, a fungal cell, an archaea cell, a plant cell or an animal cell, such as a mammalian cell. In some embodiments, this may be an organ or an animal organism. In some embodiments, the cell may be any cell type or cell line, including but not limited to, 293-T cells, 3T3 cells, 721 cells, 9 L cells, A2780 cells, A2780ADR cells, A2780cis cells, A172 cells, A20 cells, A253 cells, A431 cells, A-549 cells, ALC cells, B16 cells, B35 cells, BCP-1 cells, BEAS-2B cells, bEnd.3 cells, BHK-21 cells, BR 293 cells, BxPC3 cells, C2C12 cells, C3H-10T1/2 cells, C6/36 cells, Cal-27 cells, CHO cells, COR-L23 cells, COR-L23/CPR cells, COR-L23/5010 cells, COR-L23/R23 cells, COS-7 cells, COV-434 cells, CML T1 cells, CMT cells, CT26 cells, D17 cells, DH82 cells, DU145 cells, DuCaP cells, EL4 cells, EM2 cells, EM3 cells, EMT6/AR1 cells, EMT6/AR10.0 cells, FM3 cells, H1299 cells, H69 cells, HB54 cells, HB55 cells, HCA2 cells, HEK-293 cells, HeLa cells, Hepalclc7 cells, HL-60 cells, HMEC cells, HT-29 cells, Jurkat cells, J558L cells, JY cells, K562 cells, Ku812 cells, KCL22 cells, KG1 cells, KYO1 cells, LNCap cells, Ma-Mel 1, 2, 3 . . . 48 cells, MC-38 cells, MCF-7 cells, MCF-10A cells, MDA-MB-231 cells, MDA-MB-468 cells, MDA-MB-435 cells, MDCK II cells, MDCK II cells, MG63 cells, MOR/0.2R cells, MONO-MAC 6 cells, MRC5 cells, MTD-1A cells, MyEnd cells, NCI-H69/CPR cells, NCI-H69/LX10 cells, NCI-H69/LX20 cells, NCI-H69/LX4 cells, NIH-3T3 cells, NALM-1 cells, NW-145 cells, OPCN/OPCT cells, Peer cells, PNT-1A/PNT 2 cells, Raji cells, RBL cells, RenCa cells, RIN-5F cells, RMA/RMAS cells, Saos-2 cells, Sf-9 cells, SiHa cells, SkBr3 cells, T2 cells, T-47D cells, T84 cells, THP1 cells, U373 cells, U87 cells, U937 cells, VCaP cells, Vero cells, WM39 cells, WT-49 cells, X63 cells, YAC-1 cells, YAR cells, GM12878, K562, H1 human embryonic stem cells, HeLa-S3, HepG2, HUVEC, SK-N-SH, IMR90, A549, MCF7, HMEC or LHCM, CD14+, CD20+, primary heart or liver cells, differentiated H1 cells, 8988T, Adult_CD4_naive, Adult_CD4_Th0, Adult_CD4_Th1, AG04449, AG04450, AG09309, AG09319, AG10803, AoAF, AoSMC, BC_Adipose_UHN00001, BC_Adrenal_Gland_H12803N, BC_Bladder_01-11002, BC_Brain_H11058N, BC_Breast_02-03015, BC_Colon_01-11002, BC_Colon_H12817N, BC_Esophagus_01-11002, BC_Esophagus_H12817N, BC_Jejunum_H12817N, BC_Kidney_O1-11002, BC_Kidney_H12817N, BC_Left_Ventricle_N41, BC_Leukocyte_UHN00204, BC_Liver_01-11002, BC_Lung_01-11002, BC_Lung_H12817N, BC_Pancreas_H12817N, BC_Penis_H12817N, BC_Pericardium_H12529N, BC_Placenta_UHN00189, BC_Prostate_Gland_H12817N, BC_Rectum_N29, BC_Skeletal_Muscle_01-11002, BC_Skeletal_Muscle_H12817N, BC_Skin_01-11002, BC_Small_Intestine_01-11002, BC_Spleen_H12817N, BC_Stomach_01-11002, BC_Stomach_H12817N, BC_Testis_N30, BC_Uterus_BN0765, BE2_C, BG02ES, BG02ES-EBD, BJ, bone_marrow_HS27a, bone_marrow_HS5, bone_marrow_MSC, Breast_OC, Caco-2, CD20+_RO01778, CD20+_RO01794, CD34+_Mobilized, CD4+_Naive_Wb11970640, CD4+_Naive_Wb78495824, Cerebellum_OC, Cerebrum_frontal_OC, Chorion, CLL, CMK, Colo829, Colon_BC, Colon_OC, Cord_CD4_naive, Cord_CD4_Th0, Cord_CD4_Th1, Decidua, Dnd41, ECC-1, Endometrium_OC, Esophagus_BC, Fibrobl, Fibrobl_GM03348, FibroP, FibroP_AG08395, FibroP_AG08396, FibroP_AG20443, Frontal_cortex_OC, GC_B_cell, Gliobla, GM04503, GM04504, GM06990, GM08714, GM10248, GM10266, GM10847, GM12801, GM12812, GM12813, GM12864, GM12865, GM12866, GM12867, GM12868, GM12869, GM12870, GM12871, GM12872, GM12873, GM12874, GM12875, GM12878-XiMat, GM12891, GM12892, GM13976, GM13977, GM15510, GM18505, GM18507, GM18526, GM18951, GM19099, GM19193, GM19238, GM19239, GM19240, GM20000, H0287, H1-neurons, H7-hESC, H9ES, H9ES-AFP−, H9ES-AFP+, H9ES-CM, H9ES-E, H9ES-EB, H9ES-EBD, HAc, HAEpiC, HA-h, HAL, HAoAF, HAoAF_6090101.11, HAoAF_6111301.9, HAoEC, HAoEC_7071706.1, HAoEC_8061102.1, HA-sp, HBMEC, HBVP, HBVSMC, HCF, HCFaa, HCH, HCH_0011308.2P, HCH_8100808.2, HCM, HConF, HCPEpiC, HCT-116, Heart_OC, Heart_STL003, HEEpiC, HEK293, HEK293T, HEK293-T-REx, Hepatocytes, HFDPC, HFDPC_0100503.2, HFDPC_0102703.3, HFF, HFF-Myc, HFL11W, HFL24W, HGF, HHSEC, HIPEpiC, HL-60, HMEpC, HMEpC_6022801.3, HMF, hMNC-CB, hMNC-CB_8072802.6, hMNC-CB_9111701.6, hMNC-PB, hMNC-PB_0022330.9, hMNC-PB_0082430.9, hMSC-AT, hMSC-AT_0102604.12, hMSC-AT_9061601.12, hMSC-BM, hMSC-BM_0050602.11, hMSC-BM_0051105.11, hMSC-UC, hMSC-UC_0052501.7, hMSC-UC_0081101.7, HMVEC-dAd, HMVEC-dBl-Ad, HMVEC-dBl-Neo, HMVEC-dLy-Ad, HMVEC-dLy-Neo, HMVEC-dNeo, HMVEC-LBI, HMVEC-LLy, HNPCEpiC, HOB, HOB_0090202.1, HOB_0091301, HPAEC, HPAEpiC, HPAF, HPC-PL, HPC-PL_0032601.13, HPC-PL_0101504.13, HPDE6-E6E7, HPdLF, HPF, HPIEpC, HPIEpC_9012801.2, HPIEpC_9041503.2, HRCEpiC, HRE, HRGEC, HRPEpiC, HSaVEC, HSaVEC_0022202.16, HSaVEC_9100101.15, HSMM, HSMM_emb, HSMM_FSHD, HSMMtube, HSMMtube_emb, HSMMtube_FSHD, HT-1080, HTR8svn, Huh-7, Huh-7.5, HVMF, HVMF_6091203.3, HVMF_6100401.3, HWP, HWP_0092205, HWP_8120201.5, iPS, iPS_CWRU1, iPS_hFib2_iPS4, iPS_hFib2_iPS5, iPS NIHil 1, iPS_NIHi7, Ishikawa, Jurkat, Kidney BC, Kidney_OC, LHCN-M2, LHSR, Liver_OC, Liver_STL004, Liver_STL011, LNCaP, Loucy, Lung_BC, Lung_OC, Lymphoblastoid_cell_line, M059J, MCF10A-Er-Src, MCF-7, MDA-MB-231, Medullo, Medullo_D341, Mel 2183, Melano, Monocytes-CD14+, Monocytes-CD14+_RO01746, Monocytes-CD14+_RO01826, MRT_A204, MRT_G401, MRT_TTC549, Myometr, Naive_B_cell, NB4, NH-A, NHBE, NHBE_RA, NHDF, NHDF_0060801.3, NHDF_7071701.2, NHDF-Ad, NHDF-neo, NHEK, NHEM.f_M2, NHEM.f_M2_5071302.2, NHEM.f_M2_6022001, NHEM_M2, NHEM_M2_7011001.2, NHEM_M2_7012303, NHLF, NT2-D1, Olf_neurosphere, Osteobl, ovcar-3, PANC-1, Pancreas_OC, PanIsletD, PanIslets, PBDE, PBDEFetal, PBMC, PFSK-1, pHTE, Pons_OC, PrEC, ProgFib, Prostate, Prostate_OC, Psoas_muscle_OC, Raji, RCC_7860, RPMI-7951, RPTEC, RWPE1, SAEC, SH-SY5Y, Skeletal_Muscle_BC, SkMC, SKMC, SkMC_8121902.17, SkMC_9011302, SK-N-MC, SK-N-SH_RA, Small_intestine_OC, Spleen_OC, Stellate, Stomach_BC, T_cells_CD4+, T-47D, T98G, TBEC, Th1, Th1_Wb33676984, Th1_Wb54553204, Th17, Th2, Th2_Wb33676984, Th2_Wb54553204, Treg_Wb78495824, Treg_Wb83319432, U20S, U87, UCH-1, Urothelia, WERI-Rb-1, and WI-38. In some embodiments, the target cell can be any cell, such as a primary cell, a HEK293 cell, 293 Ts cell, SKBR3 cell, A431 cell, K562 cell, HCT116 cell, HepG2 cell, or K-Ras-dependent and K-Ras-independent cell groups.

10. METHODS OF EPIGENOMIC EDITING

The present disclosure relates to a method of epigenomic editing in a target cell or a subject with a CRISPR/Cas9-based system or CRISPR/Cpf1-based system. The method can be used to activate or repress a target gene. The method includes contacting a cell or a subject with an effective amount of the optimized gRNA molecule, as described herein, and a CRISPR/Cas9-based system or CRISPR/Cpf1-based system. In some embodiments, the optimized gRNA is encoded by a polynucleotide sequence and packaged into a lentiviral vector. In some embodiments, the lentiviral vector comprises an expression cassette comprising a promoter operably linked to the polynucleotide sequence encoding the sgRNA. In some embodiments, the promoter operably linked to the polynucleotide encoding the optimized gRNA is inducible.

11. METHODS OF SITE-SPECIFIC DNA CLEAVAGE

The present disclosure relates to a method of site specific DNA cleavage in a target cell or a subject with a CRISPR/Cas9-based system or CRISPR/Cpf1-based system. The method includes contacting a cell or a subject with an effective amount of the optimized gRNA molecule, as described herein, and a CRISPR/Cas9-based system or CRISPR/Cpf1-based system. In some embodiments, the optimized gRNA is encoded by a polynucleotide sequence and packaged into a lentiviral vector. In some embodiments, the lentiviral vector comprises an expression cassette comprising a promoter operably linked to the polynucleotide sequence encoding the sgRNA. In some embodiments, the promoter operably linked to the polynucleotide encoding the optimized gRNA is inducible.

The number of gRNA administered to the cell or sample may be at least 1 gRNA, at least 2 different gRNA, at least 3 different gRNA at least 4 different gRNA, at least 5 different gRNA, at least 6 different gRNA, at least 7 different gRNA, at least 8 different gRNA, at least 9 different gRNA, at least 10 different gRNAs, at least 11 different gRNAs, at least 12 different gRNAs, at least 13 different gRNAs, at least 14 different gRNAs, at least 15 different gRNAs, at least 16 different gRNAs, at least 17 different gRNAs, at least 18 different gRNAs, at least 18 different gRNAs, at least 20 different gRNAs, at least 25 different gRNAs, at least 30 different gRNAs, at least 35 different gRNAs, at least 40 different gRNAs, at least 45 different gRNAs, or at least 50 different gRNAs. The number of gRNA administered to the cell may be between at least 1 gRNA to at least 50 different gRNAs, at least 1 gRNA to at least 45 different gRNAs, at least 1 gRNA to at least 40 different gRNAs, at least 1 gRNA to at least 35 different gRNAs, at least 1 gRNA to at least 30 different gRNAs, at least 1 gRNA to at least 25 different gRNAs, at least 1 gRNA to at least 20 different gRNAs, at least 1 gRNA to at least 16 different gRNAs, at least 1 gRNA to at least 12 different gRNAs, at least 1 gRNA to at least 8 different gRNAs, at least 1 gRNA to at least 4 different gRNAs, at least 4 gRNAs to at least 50 different gRNAs, at least 4 different gRNAs to at least 45 different gRNAs, at least 4 different gRNAs to at least 40 different gRNAs, at least 4 different gRNAs to at least 35 different gRNAs, at least 4 different gRNAs to at least 30 different gRNAs, at least 4 different gRNAs to at least 25 different gRNAs, at least 4 different gRNAs to at least 20 different gRNAs, at least 4 different gRNAs to at least 16 different gRNAs, at least 4 different gRNAs to at least 12 different gRNAs, at least 4 different gRNAs to at least 8 different gRNAs, at least 8 different gRNAs to at least 50 different gRNAs, at least 8 different gRNAs to at least 45 different gRNAs, at least 8 different gRNAs to at least 40 different gRNAs, at least 8 different gRNAs to at least 35 different gRNAs, 8 different gRNAs to at least 30 different gRNAs, at least 8 different gRNAs to at least 25 different gRNAs, 8 different gRNAs to at least 20 different gRNAs, at least 8 different gRNAs to at least 16 different gRNAs, or 8 different gRNAs to at least 12 different gRNAs.

The gRNA may comprise a complementary polynucleotide sequence of the target DNA sequence followed by a PAM sequence. The gRNA may comprise a “G” at the 5′ end of the complementary polynucleotide sequence. The gRNA may comprise at least a 10 base pair, at least a 11 base pair, at least a 12 base pair, at least a 13 base pair, at least a 14 base pair, at least a 15 base pair, at least a 16 base pair, at least a 17 base pair, at least a 18 base pair, at least a 19 base pair, at least a 20 base pair, at least a 21 base pair, at least a 22 base pair, at least a 23 base pair, at least a 24 base pair, at least a 25 base pair, at least a 30 base pair, or at least a 35 base pair complementary polynucleotide sequence of the target DNA sequence followed by a PAM sequence. The PAM sequence may be “NGG”, where “N” can be any nucleotide. The gRNA may target at least one of the promoter region, the enhancer region or the transcribed region of the target gene. In some embodiments, the gRNA targets a nucleic acid sequence having a polynucleotide sequence of at least one of SEQ ID NOs: 13-148, 316, 317, or 320. The gRNA may include a nucleic acid sequence of at least one of SEQ ID NOs: 149-315, 321-323, or 326-329.

12. METHODS OF CORRECTING A MUTANT GENE AND TREATING A SUBJECT

The present disclosure is also directed to a method of correcting a mutant gene in a subject. The method comprises administering to a cell of the subject the composition, as described above. Use of the composition to deliver the CRISPR/Cas9-based system or CRISPR/Cpf1-based system with at least one gRNA, such as an optimized gRNA described herein, to the cell may restore the expression of a full-functional or partially-functional protein with a repair template or donor DNA, which can replace the entire gene or the region containing the mutation. The CRISPR/Cas9-based system or CRISPR/Cpf1-based system with at least one gRNA, such as an optimized gRNA described herein, may be used to introduce site-specific double strand breaks at targeted genomic loci. Site-specific double-strand breaks are created when the CRISPR/Cas9-based system or CRISPR/Cpf1-based system with at least one gRNA, such as an optimized gRNA described herein, binds to a target DNA sequences, thereby permitting cleavage of the target DNA. This DNA cleavage may stimulate the natural DNA-repair machinery, leading to one of two possible repair pathways: homology-directed repair (HDR) or the non-homologous end joining (NHEJ) pathway.

The present disclosure is directed to genome editing with a CRISPR/Cas9-based system or CRISPR/Cpf1-based system with at least one gRNA, such as an optimized gRNA described herein, without a repair template, which can efficiently correct the reading frame and restore the expression of a functional protein involved in a genetic disease. The disclosed CRISPR/Cas9-based system or CRISPR/Cpf1-based system with at least one gRNA, such as an optimized gRNA described herein, may involve using homology-directed repair or nuclease-mediated non-homologous end joining (NHEJ)-based correction approaches, which enable efficient correction in proliferation-limited primary cell lines that may not be amenable to homologous recombination or selection-based gene correction. This strategy integrates the rapid and robust assembly of active the CRISPR/Cas9-based system or CRISPR/Cpf1-based system with at least one gRNA, such as an optimized gRNA described herein, with an efficient gene editing method for the treatment of genetic diseases caused by mutations in nonessential coding regions that cause frameshifts, premature stop codons, aberrant splice donor sites or aberrant splice acceptor sites.

a. Nuclease Mediated Non-Homologous End Joining

Restoration of protein expression from an endogenous mutated gene may be through template-free NHEJ-mediated DNA repair. In contrast to a transient method targeting the target gene RNA, the correction of the target gene reading frame in the genome by a transiently expressed CRISPR/Cas9-based system or CRISPR/Cpf1-based system with at least one gRNA, such as an optimized gRNA described herein, may lead to permanently restored target gene expression by each modified cell and all of its progeny.

Nuclease mediated NHEJ gene correction may correct the mutated target gene and offers several potential advantages over the HDR pathway. For example, NHEJ does not require a donor template, which may cause nonspecific insertional mutagenesis. In contrast to HDR, NHEJ operates efficiently in all stages of the cell cycle and therefore may be effectively exploited in both cycling and post-mitotic cells, such as muscle fibers. This provides a robust, permanent gene restoration alternative to oligonucleotide-based exon skipping or pharmacologic forced read-through of stop codons and could theoretically require as few as one drug treatment. NHEJ-based gene correction using a CRISPR/Cas9-based system or CRISPR/Cpf1-based system, as well as other engineered nucleases including meganucleases and zinc finger nucleases, may be combined with other existing ex vivo and in vivo platforms for cell- and gene-based therapies, in addition to the plasmid electroporation approach described here. For example, delivery of a CRISPR/Cas9-based system or CRISPR/Cpf1-based system by mRNA-based gene transfer or as purified cell permeable proteins could enable a DNA-free genome editing approach that would circumvent any possibility of insertional mutagenesis.

b. Homology-Directed Repair

Restoration of protein expression from an endogenous mutated gene may involve homology-directed repair. The method as described above further includes administrating a donor template to the cell. The donor template may include a nucleotide sequence encoding a full-functional protein or a partially-functional protein. For example, the donor template may include a miniaturized dystrophin construct, termed minidystrophin (“minidys”), a full-functional dystrophin construct for restoring a mutant dystrophin gene, or a fragment of the dystrophin gene that after homology-directed repair leads to restoration of the mutant dystrophin gene.

13. METHODS OF GENOME EDITING

The present disclosure is also directed to genome editing with the CRISPR/Cas9-based system or CRISPR/Cpf1-based system described above to restore the expression of a full-functional or partially-functional protein with a repair template or donor DNA, which can replace the entire gene or the region containing the mutation. The CRISPR/Cas9-based system or CRISPR/Cpf1-based system may be used to introduce site-specific double strand breaks at targeted genomic loci. Site-specific double-strand breaks are created when the CRISPR/Cas9-based system or CRISPR/Cpf1-based system binds to a target DNA sequences using the gRNA, thereby permitting cleavage of the target DNA. The CRISPR/Cas9-based system and CRISPR/Cpf1-based system has the advantage of advanced genome editing due to their high rate of successful and efficient genetic modification. This DNA cleavage may stimulate the natural DNA-repair machinery, leading to one of two possible repair pathways: homology-directed repair (HDR) or the non-homologous end joining (NHEJ) pathway.

The present disclosure is directed to genome editing with CRISPR/Cas9-based system or CRISPR/Cpf1-based system without a repair template, which can efficiently correct the reading frame and restore the expression of a functional protein involved in a genetic disease. The disclosed CRISPR/Cas9-based system or CRISPR/Cpf1-based system and methods may involve using homology-directed repair or nuclease-mediated non-homologous end joining (NHEJ)-based correction approaches, which enable efficient correction in proliferation-limited primary cell lines that may not be amenable to homologous recombination or selection-based gene correction. This strategy integrates the rapid and robust assembly of active CRISPR/Cas9-based system or CRISPR/Cpf1-based system with an efficient gene editing method for the treatment of genetic diseases caused by mutations in nonessential coding regions that cause frameshifts, premature stop codons, aberrant splice donor sites or aberrant splice acceptor sites.

The present disclosure provides methods of correcting a mutant gene in a cell and treating a subject suffering from a genetic disease, such as DMD. The method may include administering to a cell or subject a CRISPR/Cas9-based system or CRISPR/Cpf1-based system, a polynucleotide or vector encoding said CRISPR/Cas9-based system or CRISPR/Cpf1-based system, or composition of said CRISPR/Cas9-based system or CRISPR/Cpf1-based system as described above. The method may include administering a CRISPR/Cas9-based system or CRISPR/Cpf1-based system, such as administering a Cas9 protein, a Cpf1 protein, a Cas9 fusion protein containing a second domain, a nucleotide sequence encoding said Cas9 protein, Cpf1 protein, or Cas9 fusion protein, and/or at least one gRNA, wherein the gRNAs target different DNA sequences. The target DNA sequences may be overlapping. The number of gRNA administered to the cell may be at least 1 gRNA, at least 2 different gRNA, at least 3 different gRNA at least 4 different gRNA, at least 5 different gRNA, at least 6 different gRNA, at least 7 different gRNA, at least 8 different gRNA, at least 9 different gRNA, at least 10 different gRNA, at least 15 different gRNA, at least 20 different gRNA, at least 30 different gRNA, or at least 50 different gRNA, as described above. The gRNA may include a nucleic acid sequence of at least one of SEQ ID NOs: 149-315, 321-323, or 326-329. The method may involve homology-directed repair or non-homologous end joining.

14. CONSTRUCTS AND PLASMIDS

The compositions, as described above, may comprise genetic constructs that encodes the CRISPR/Cas9-based system or CRISPR/Cpf1-based system, as disclosed herein. The genetic construct, such as a plasmid, may comprise a nucleic acid that encodes the CRISPR/Cas9-based system or CRISPR/Cpf1-based system, such as the Cas9 protein, the Cpf1 protein, and Cas9 fusion proteins and/or at least one of the optimized gRNAs as described herein. The compositions, as described above, may comprise genetic constructs that encodes the modified AAV vector and a nucleic acid sequence that encodes the CRISPR/Cas9-based system or CRISPR/Cpf1-based system with at least one gRNA, such as an optimized gRNA described herein. The genetic construct, such as a plasmid, may comprise a nucleic acid that encodes the CRISPR/Cas9-based system or CRISPR/Cpf1-based system with at least one gRNA, such as an optimized gRNA described herein. The compositions, as described above, may comprise genetic constructs that encodes the modified lentiviral vector, as disclosed herein. The genetic construct, such as a plasmid, may comprise a nucleic acid that encodes a Cas9-fusion protein and at least one sgRNA. The genetic construct may be present in the cell as a functioning extrachromosomal molecule. The genetic construct may be a linear minichromosome including centromere, telomeres or plasmids or cosmids.

The genetic construct may also be part of a genome of a recombinant viral vector, including recombinant lentivirus, recombinant adenovirus, and recombinant adenovirus associated virus. The genetic construct may be part of the genetic material in attenuated live microorganisms or recombinant microbial vectors which live in cells. The genetic constructs may comprise regulatory elements for gene expression of the coding sequences of the nucleic acid. The regulatory elements may be a promoter, an enhancer, an initiation codon, a stop codon, or a polyadenylation signal.

The nucleic acid sequences may make up a genetic construct that may be a vector. The vector may be capable of expressing the fusion protein, such as a Cas9-fusion protein, in the cell of a mammal. The vector may be recombinant. The vector may comprise heterologous nucleic acid encoding the Cas9-fusion protein. The vector may be a plasmid. The vector may be useful for transfecting cells with nucleic acid encoding the Cas9-fusion protein, which the transformed host cell is cultured and maintained under conditions wherein expression of the Cas9-fusion protein system takes place.

Coding sequences may be optimized for stability and high levels of expression. In some instances, codons are selected to reduce secondary structure formation of the RNA such as that formed due to intramolecular bonding.

The vector may comprise heterologous nucleic acid encoding the CRISPR/Cas9-based system or CRISPR/Cpf1-based system and may further comprise an initiation codon, which may be upstream of the CRISPR/Cas9-based system or CRISPR/Cpf1-based system coding sequence, and a stop codon, which may be downstream of the CRISPR/Cas9-based system or CRISPR/Cpf1-based system coding sequence. The initiation and termination codon may be in frame with the CRISPR/Cas9-based system or CRISPR/Cpf1-based system coding sequence. The vector may also comprise a promoter that is operably linked to the CRISPR/Cas9-based system or CRISPR/Cpf1-based system coding sequence. The promoter operably linked to the CRISPR/Cas9-based system or CRISPR/Cpf1-based system coding sequence may be a promoter from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter may also be a promoter from a human gene such as human ubiquitin C (hUbC), human actin, human myosin, human hemoglobin, human muscle creatine, or human metalothionein. The promoter may also be a tissue specific promoter, such as a muscle or skin specific promoter, natural or synthetic. Examples of such promoters are described in US Patent Application Publication No. US20040175727, the contents of which are incorporated herein in its entirety.

The vector may also comprise a polyadenylation signal, which may be downstream of the CRISPR/Cas9-based system or CRISPR/Cpf1-based system. The polyadenylation signal may be a SV40 polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, or human β-globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal from a pCEP4 vector (Invitrogen, San Diego, Calif.).

The vector may also comprise an enhancer upstream of the CRISPR/Cas9-based system or CRISPR/Cpf1-based system, i.e., the Cas9 protein, the Cpf1 protein, or Cas9 fusion protein coding sequence or sgRNA, such as an optimized gRNA described herein. The enhancer may be necessary for DNA expression. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine or a viral enhancer such as one from CMV, HA, RSV or EBV. Polynucleotide function enhancers are described in U.S. Pat. Nos. 5,593,972, 5,962,428, and WO94/016737, the contents of each are fully incorporated by reference. The vector may also comprise a mammalian origin of replication in order to maintain the vector extrachromosomally and produce multiple copies of the vector in a cell. The vector may also comprise a regulatory sequence, which may be well suited for gene expression in a mammalian or human cell into which the vector is administered. The vector may also comprise a reporter gene, such as green fluorescent protein (“GFP”) and/or a selectable marker, such as hygromycin (“Hygro”).

The vector may be expression vectors or systems to produce protein by routine techniques and readily available starting materials including Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989), which is incorporated fully by reference. In some embodiments the vector may comprise the nucleic acid sequence encoding the CRISPR/Cas9-based system or CRISPR/Cpf1-based system, including the nucleic acid sequence encoding the Cas9 protein, the Cpf1 protein, or Cas9 fusion protein and the nucleic acid sequence encoding the at least one gRNA comprising the nucleic acid sequence of at least one of SEQ ID NOs: 149-315, 321-323, or 326-329.

15. PHARMACEUTICAL COMPOSITIONS

The composition may be in a pharmaceutical composition. The pharmaceutical composition may comprise about 1 ng to about 10 mg of DNA encoding the CRISPR/Cas9-based system, CRISPR/Cpf1-based system, or CRISPR/Cas9-based system protein component, i.e., the Cas9 protein, the Cpf1 protein, or Cas9 fusion protein. The pharmaceutical composition may comprise about 1 ng to about 10 mg of the DNA of the modified AAV vector and nucleotide sequence encoding the CRISPR/Cas9-based system with at least one gRNA, such as an optimized gRNA described herein. The pharmaceutical composition may comprise about 1 ng to about 10 mg of the DNA of the modified lentiviral vector. The pharmaceutical compositions according to the present invention are formulated according to the mode of administration to be used. In cases where pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, pyrogen free and particulate free. An isotonic formulation is preferably used. Generally, additives for isotonicity may include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstriction agent is added to the formulation.

The composition may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be functional molecules as vehicles, adjuvants, carriers, or diluents. The pharmaceutically acceptable excipient may be a transfection facilitating agent, which may include surface active agents, such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents.

The transfection facilitating agent is a polyanion, polycation, including poly-L-glutamate (LGS), or lipid. The transfection facilitating agent is poly-L-glutamate, and more preferably, the poly-L-glutamate is present in the composition for genome editing at a concentration less than 6 mg/ml. The transfection facilitating agent may also include surface active agents such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid may also be used administered in conjunction with the genetic construct. In some embodiments, the DNA vector encoding the composition may also include a transfection facilitating agent such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art, as a DNA-liposome mixture (see for example WO9324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. Preferably, the transfection facilitating agent is a polyanion, polycation, including poly-L-glutamate (LGS), or lipid.

16. CONSTRUCTS AND PLASMIDS

The compositions, as described above, may comprise genetic constructs that encodes the CRISPR/Cas9-based system or CRISPR/Cpf1-based system, as disclosed herein. The genetic construct, such as a plasmid or expression vector, may comprise a nucleic acid that encodes the CRISPR/Cas9-based system or CRISPR/Cpf1-based system, and/or at least one gRNA, such as an optimized gRNA described herein. The compositions, as described above, may comprise genetic constructs that encodes the modified lentiviral vector and a nucleic acid sequence that encodes the CRISPR/Cas9-based system or CRISPR/Cpf1-based system, as disclosed herein. The genetic construct, such as a plasmid, may comprise a nucleic acid that encodes the CRISPR/Cas9-based system or CRISPR/Cpf1-based system. The compositions, as described above, may comprise genetic constructs that encodes a modified lentiviral vector. The genetic construct, such as a plasmid, may comprise a nucleic acid that encodes the CRISPR/Cas9-based system or CRISPR/Cpf1-based system and at least one sgRNA such as an optimized gRNA described herein. The genetic construct may be present in the cell as a functioning extrachromosomal molecule. The genetic construct may be a linear minichromosome including centromere, telomeres or plasmids or cosmids.

The genetic construct may also be part of a genome of a recombinant viral vector, including recombinant lentivirus, recombinant adenovirus, and recombinant adenovirus associated virus. The genetic construct may be part of the genetic material in attenuated live microorganisms or recombinant microbial vectors which live in cells. The genetic constructs may comprise regulatory elements for gene expression of the coding sequences of the nucleic acid. The regulatory elements may be a promoter, an enhancer, an initiation codon, a stop codon, or a polyadenylation signal.

The nucleic acid sequences may make up a genetic construct that may be a vector. The vector may be capable of expressing the fusion protein, such as the CRISPR/Cas9-based system or CRISPR/Cpf1-based system, in the cell of a mammal. The vector may be recombinant. The vector may comprise heterologous nucleic acid encoding the fusion protein, such as the CRISPR/Cas9-based system. The vector may be a plasmid. The vector may be useful for transfecting cells with nucleic acid encoding the CRISPR/Cas9-based system or CRISPR/Cpf1-based system, which the transformed host cell is cultured and maintained under conditions wherein expression of the CRISPR/Cas9-based system or CRISPR/Cpf1-based system takes place.

Coding sequences may be optimized for stability and high levels of expression. In some instances, codons are selected to reduce secondary structure formation of the RNA such as that formed due to intramolecular bonding.

The vector may comprise heterologous nucleic acid encoding the CRISPR/Cas9-based system or CRISPR/Cpf1-based system and may further comprise an initiation codon, which may be upstream of the CRISPR/Cas9-based system or CRISPR/Cpf1-based system coding sequence, and a stop codon, which may be downstream of the CRISPR/Cas9-based system or CRISPR/Cpf1-based system coding sequence. The initiation and termination codon may be in frame with the CRISPR/Cas9-based system or CRISPR/Cpf1-based system coding sequence. The vector may also comprise a promoter that is operably linked to the CRISPR/Cas9-based system or CRISPR/Cpf1-based system coding sequence. The CRISPR/Cas9-based system or CRISPR/Cpf1-based system may be under the light-inducible or chemically inducible control to enable the dynamic control of in space and time. The promoter operably linked to the CRISPR/Cas9-based system or CRISPR/Cpf1-based system coding sequence may be a promoter from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter may also be a promoter from a human gene such as human ubiquitin C (hUbC), human actin, human myosin, human hemoglobin, human muscle creatine, or human metalothionein. The promoter may also be a tissue specific promoter, such as a muscle or skin specific promoter, natural or synthetic. Examples of such promoters are described in US Patent Application Publication No. US20040175727, the contents of which are incorporated herein in its entirety.

The vector may also comprise a polyadenylation signal, which may be downstream of the CRISPR/Cas9-based system or CRISPR/Cpf1-based system. The polyadenylation signal may be a SV40 polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, or human β-globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal from a pCEP4 vector (Invitrogen, San Diego, Calif.).

The vector may also comprise an enhancer upstream of the CRISPR/Cas9-based system or CRISPR/Cpf1-based system and/or sgRNA, such as an optimized gRNA described herein. The enhancer may be necessary for DNA expression. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine or a viral enhancer such as one from CMV, HA, RSV or EBV. Polynucleotide function enhancers are described in U.S. Pat. Nos. 5,593,972, 5,962,428, and WO94/016737, the contents of each are fully incorporated by reference. The vector may also comprise a mammalian origin of replication in order to maintain the vector extrachromosomally and produce multiple copies of the vector in a cell. The vector may also comprise a regulatory sequence, which may be well suited for gene expression in a mammalian or human cell into which the vector is administered. The vector may also comprise a reporter gene, such as green fluorescent protein (“GFP”) and/or a selectable marker, such as hygromycin (“Hygro”).

The vector may be expression vectors or systems to produce protein by routine techniques and readily available starting materials including Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989), which is incorporated fully by reference. In some embodiments the vector may comprise the nucleic acid sequence encoding the CRISPR/Cas9-based system or CRISPR/Cpf1-based system and the nucleic acid sequence encoding the at least one gRNA, such as an optimized gRNA described herein.

In some embodiments, the gRNA, such as an optimized gRNA described herein, is encoded by a polynucleotide sequence and packaged into a lentiviral vector. In some embodiments, the lentiviral vector includes an expression cassette. The expression cassette can includes a promoter operably linked to the polynucleotide sequence encoding the gRNA, such as an optimized gRNA described herein. In some embodiments, the promoter operably linked to the polynucleotide encoding the gRNA is inducible.

i. Adeno-Associated Virus Vectors

The composition, as described above, includes a modified adeno-associated virus (AAV) vector. The modified AAV vector may have enhanced cardiac and skeletal muscle tissue tropism. The modified AAV vector may be capable of delivering and expressing the CRISPR/Cas9-based system or CRISPR/Cpf1-based system with at least one gRNA, such as an optimized gRNA described herein, in the cell of a mammal. For example, the modified AAV vector may be an AAV-SASTG vector (Piacentino et al. (2012) Human Gene Therapy 23:635-646). The modified AAV vector may deliver nucleases to skeletal and cardiac muscle in vivo. The modified AAV vector may be based on one or more of several capsid types, including AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9. The modified AAV vector may be based on AAV2 pseudotype with alternative muscle-tropic AAV capsids, such as AAV2/1, AAV2/6, AAV2/7, AAV2/8, AAV2/9, AAV2.5 and AAV/SASTG vectors that efficiently transduce skeletal muscle or cardiac muscle by systemic and local delivery (Seto et al. Current Gene Therapy (2012) 12:139-151).

17. METHODS OF DELIVERY

Provided herein is a method for delivering the CRISPR/Cas9-based system or CRISPR/Cpf1-based system and the optimized gRNA described herein for providing genetic constructs and/or proteins of the CRISPR/Cas9-based system or CRISPR/Cpf1-based system. The delivery of the CRISPR/Cas9-based system or CRISPR/Cpf1-based system and the optimized gRNA described herein may be the transfection or electroporation of the CRISPR/Cas9-based system or CRISPR/Cpf1-based system and the optimized gRNA described herein as one or more nucleic acid molecules that is expressed in the cell and delivered to the surface of the cell. The CRISPR/Cas9-based system or CRISPR/Cpf1-based system protein may be delivered to the cell. The nucleic acid molecules may be electroporated using BioRad Gene Pulser Xcell or Amaxa Nucleofector IIb devices or other electroporation device. Several different buffers may be used, including BioRad electroporation solution, Sigma phosphate-buffered saline product #D8537 (PBS), Invitrogen OptiMEM I (OM), or Amaxa Nucleofector solution V (N.V.). Transfections may include a transfection reagent, such as Lipofectamine 2000.

The vector encoding a CRISPR/Cas9-based system or CRISPR/Cpf1-based system protein may be delivered to the modified target cell in a tissue or subject by DNA injection (also referred to as DNA vaccination) with and without in vivo electroporation, liposome mediated, nanoparticle facilitated, and/or recombinant vectors. The recombinant vector may be delivered by any viral mode. The viral mode may be recombinant lentivirus, recombinant adenovirus, and/or recombinant adeno-associated virus.

The nucleotide encoding a CRISPR/Cas9-based system or CRISPR/Cpf1-based system protein may be introduced into a cell to induce gene expression of the target gene. For example, one or more nucleotide sequences encoding the CRISPR/Cas9-based system or CRISPR/Cpf1-based system directed towards a target gene may be introduced into a mammalian cell. Upon delivery of the CRISPR/Cas9-based system or CRISPR/Cpf1-based system to the cell, and thereupon the vector into the cells of the mammal, the transfected cells will express the CRISPR/Cas9-based system or CRISPR/Cpf1-based system. The CRISPR/Cas9-based system or CRISPR/Cpf1-based system may be administered to a mammal to induce or modulate gene expression of the target gene in a mammal. The mammal may be human, non-human primate, cow, pig, sheep, goat, antelope, bison, water buffalo, bovids, deer, hedgehogs, elephants, llama, alpaca, mice, rats, or chicken, and preferably human, cow, pig, or chicken.

Methods of introducing a nucleic acid into a host cell are known in the art, and any known method can be used to introduce a nucleic acid (e.g., an expression construct) into a cell. Suitable methods include, include e.g., viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery, and the like. In some embodiments, the composition may be delivered by mRNA delivery and ribonucleoprotein (RNP) complex delivery.

18. ROUTES OF ADMINISTRATION

The compositions may be administered to a subject by different routes including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, intrapleurally, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal intrathecal, and intraarticular or combinations thereof. For veterinary use, the composition may be administered as a suitably acceptable formulation in accordance with normal veterinary practice. The veterinarian may readily determine the dosing regimen and route of administration that is most appropriate for a particular animal. The compositions may be administered by traditional syringes, needleless injection devices, “microprojectile bombardment gone guns”, or other physical methods such as electroporation (“EP”), “hydrodynamic method”, or ultrasound.

The composition may be delivered to the mammal by several technologies including DNA injection (also referred to as DNA vaccination) with and without in vivo electroporation, liposome mediated, nanoparticle facilitated, recombinant vectors such as recombinant lentivirus, recombinant adenovirus, and recombinant adenovirus associated virus. The composition may be injected into the skeletal muscle or cardiac muscle. For example, the composition may be injected into the tibialis anterior muscle.

19. KITS

Provided herein is a kit, which may be used for site-specific DNA binding. The kit comprises a composition, as described above, and instructions for using said composition. Instructions included in kits may be affixed to packaging material or may be included as a package insert. While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” may include the address of an internet site that provides the instructions.

The composition may include a modified lentiviral vector and a nucleotide sequence encoding a CRISPR/Cas9-based system and the optimized gRNA, as described above. The CRISPR/Cas9-based system, as described above, may be included in the kit to specifically bind and target a particular regulatory region of the target gene.

20. EXAMPLES

The foregoing may be better understood by reference to the following examples, which are presented for purposes of illustration and are not intended to limit the scope of the invention.

Example 1 Materials and Methods

Materials. Tris-HCl (pH 7.6) buffer was obtained from Corning Life Sciences. L-glutamic acid monopotassium salt monohydrate, dithiothreitol (DTT), and magnesium chloride were obtained from Sigma Aldrich Co., LLC.

Cloning of Cas9, dCas9, and s2RNA Expression Plasmids; Plasmids encoding Cas9, dCas9, and sgRNAs which target the AAVS1 locus of human chromosome 19 were cloned, expressed, and purified using standard techniques. The DNA substrates used for imaging-(i) a 1198 bp substrate derived from a segment of the AAVS1 locus of human chromosome 19; (ii) an ‘engineered’ 989 bp DNA substrate containing a series of six full, partial, or mismatched target sites; and (iii) a 1078 bp ‘nonsense’ substrate containing no homology to the protospacer (>3 bp)—were also generated using standard techniques. The plasmids encoding wild-type Cas9 and dCas9 were obtained from Addgene (plasmid 39312 and plasmid 47106). Plasmids for the expression of Cas9 and dCas9 in bacteria were cloned using Gateway Cloning (Life Technologies). Briefly, PCR was used to amplify Cas9 and dCas9 genes and to add flanking attL1 and attL2 sites. BP recombination was performed to transfer these genes to a shuttle vector, after which LP recombination was performed to transfer these genes to pDest17, which adds an N-terminal hexa-histidine tag (Life Technologies). The plasmids encoding the chimeric sgRNA and sgRNA variants (described below) were cloned as previously described (Perez-Pinera et al., (2013) Nature methods, 10, 973-976).

Expression and Purification of Cas9, dCas9. Plasmids encoding Cas9 or dCas9 were transformed into SoluBL21 competent cells (Genlantis) according to standard techniques (Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular cloning. Cold spring harbor laboratory press New York.). Single colonies were used to inoculate 25 mL starter cultures. 25 mL starter cultures were grown overnight and used to inoculate 1 L cultures. Inoculated 1 L cultures were grown for 5 hours at 25° C. after which the temperature was dropped to 16° C. and protein expression induced by the addition of 0.1 mM IPTG. Induced cultures were grown for another 12 hours at 16° C. Cells were harvested by centrifugation at 4000×g and stored at −80° C. for long-term storage.

Cell pellets were resuspended in 30 mL of Lysis Buffer (50 mM Tris-HCl, 500 mM NaCl, 10 mM MgCl₂, 10% v/v glycerol, 0.2% Triton-1000, and 1 mM PMSF). The cell suspension was lysed by sonication at 30% duty cycle for 5 minutes. The suspension was then centrifuged for 30 minutes at 12,000×g. The supernatant was then taken and incubated with Ni-NTA resin (Qiagen) for 30 minutes under gentle agitation. The resin was then loaded onto a column, washed with Wash Buffer (35 mM imidizole, 50 mM Tris-HCl, 500 mM NaCl, 10 mM MgCl₂, 10% v/v glycerol), and eluted with Elution Buffer (120 mM imidizole, 50 mM Tris-HCl, 500 mM NaCl, 10 mM MgCl₂, 10% v/v glycerol). Ultracel-30k centrifugal filters were then used to exchange solvents to the Storage Buffer (50 mM Tris-HCl, 500 mM NaCl, 10 mM MgCl₂, 10% v/v glycerol). The samples were then aliquoted and frozen at −80° C. Representative polyacrylamide SDS gels of purified Cas9 and dCas9 are presented in Figure S1, indicating approximately >95% purity.

Expression and purification of s2RNA and guide RNA variants. Guide RNAs were in vitro transcribed using the MEGAshortscript T7 Transcription Kit (Life Technologies. DNA templates with a T7 promoter were generated via PCR from guide RNA plasmids and reactions were set up following the manufacturer's instructions. The T7 templates for the guide RNAs with 2 nucleotides truncated from their 5′-ends (tru-gRNAs) and those with 5′ extensions that form hairpins (hp-gRNAs) were generated by PCR off of the standard gRNA plasmids. The RNA was then purified using phenol-chloroform extraction using standard techniques (Sambrook et al. (1989) Molecular cloning. Cold spring harbor laboratory press New York).

Generation of DNA substrates. Genomic DNA was extracted and purified from HEK293T cell line using the DNeasy kit (Qiagen), following the manufacturer's protocol. The AAVS1 locus was then amplified using PCR. The 1198 bp AAVS1-derived substrate was constructed via direct PCR from genomic DNA using primers from Integrated DNA Technologies (IDT): 5′-\Bt\-CCAGGATCAGTGAAACGCAC-3′ and 5′-GAGCTCTACTGGCTTCTGCG-3′, where Bt represents a biotinylation of the primer at the 5′-end. The ‘engineered’ DNA substrate, which contains a series of PAMs and full or partial protospacer sites, was ordered as two gBlock fragments each containing an EcoRI restriction site on one end. Substrates were digested, ligated together, and then enriched via PCR with primers (Integrated DNA Technologies, IDT): 5′-\Bt\-CATGACGTGCAGCAAGC-3′ and 5′-CGACGATGCGCTGAATC-3′. To construct a ‘nonsense’ substrate containing no sites exhibiting homology (greater than 3 bp) to the protospacer: a 690 bp DNA construct was synthesized (GeneScript, Inc.) containing a series of restriction sites, and an addition length of DNA from lambda DNA (New England Biolabs) was sub-cloned into the construct; the 1078 bp substrate was then PCR amplified using primers (IDT): 5′-\Bt\-GACCTGCAGGCATGCAAGCTTGG-3′ and 5′-CAGCGTCCCCGGTTGTGAATCT-3′. All DNA was gel purified, diluted to 25 nM in working buffer (20 mM Tris-HCl (pH 7.6), 100 mM potassium glutamate, 5 mM MgCl₂, and 0.4 mM DTT) and incubated with 40× excess monomeric streptavidin (Howarth et al., (2006) Nature methods, 3, 267-273) for 10 minutes prior to incubation with Cas9/dCas9.

Sodium dodecyl sulfate-polyacrylamide gels of purified Cas9 and dCas9 are presented in FIGS. 8A-8B, indicating approximately 95% purity.

Atomic Force Microscopy. Atomic force microscopy (AFM) was performed in air using a Bruker (nee Veeco) Nanoscope V Multimode with RTSEP (Bruker) probes (nominal spring constant 40 N/m, resonance frequency, 300 kHz). Prior to experiments, protein and guide RNAs were mixed in 1:1.5 ratio for 10 minutes. Protein and DNA were mixed in a solution of working buffer for at least 10 minutes (up to 35 minutes) at room temperature, deposited for 8 seconds on freshly cleaved mica (Ted Pella, Inc.) that had been treated with 3-aminopropylsiloxane (prepared as previously described (24)), rinsed with ultra-pure (>17 MΩ) water, and dried in air. Proteins were centrifuged briefly prior to incubation with DNA. When the standard sgRNA was used, at least four preparations for each experimental condition were imaged, and at least two for experiments with the other guide RNA variants. In general images were acquired with pixel resolution of 1024×1024 over 2.75 micron square areas or 2048×2048 over 5.5 micron square areas at 1-1.5 line/s for each sample. Images of several thousand (˜2500-6000) DNA molecules were resolved for each experimental condition.

DNA Tracing and Refinement with Sub-Pixel Resolution. Acquired AFM images were flattened and leveled (plane-wise, by line, and by 3^(rd) order polynomial leveling) using an open-source image analysis software for scanning probe microscopy, Gwyddion (http://gwyddion.net/), and then exported to MATLAB (Mathworks, Inc.). 151×151 pixel (405 nm×405 nm) regions containing each DNA molecule were sorted by inspection for a clearly identifiable streptavidin label, the presence of at least one bound Cas9/dCas9 molecule, and an unambiguous end-to-end path to ensure lack of aggregation or overlap with other DNA molecules. The contour of the DNA was traced by hand and the estimated boundaries of the streptavidin and Cas9/dCas9 were marked. The trace was then algorithmically refined using a method based on Wiggins et al. (2006) Nature nanotechnology, 1, 137-141. Starting at the weighted centroid of the streptavidin (x₁), the position of next element of the backbone (x₂) is estimated by stepping 2.5 nm toward the nearest hand-traced points beyond the estimated boundary of the streptavidin. An 11-pixel line is drawn on a two-fold linear interpolation of the image of the DNA perpendicularly to the (x₁-x₂) line segment at x₂. x₂ is relocated to the position on the normal line with the maximum topographical height then adjusted to the 2.5 nm from x₁ on the new (x₁-x₂) line. The positions of x₃ . . . x_(n) are then iteratively estimated using the nearest hand-traced points to generate the initial guess for the next backbone position then corrected as before, and the correction process continues until the point x_(n) is less than 2.5 nm from the end of the traced DNA molecule. When the refined trace enters the estimated boundary of a Cas9/dCas9 molecule at x_(i), the position of the DNA is instead estimated as the point on a cubic Hermite spline (using points x_(i−1), x_(i), x_(j), and x_(j+1), where x_(j) is the first point of the hand-drawn trace beyond the estimated Cas9/dCas9 boundary) located 2.5 nm from x_(i).

Upon completion of the trace, the height of the DNA along the contour is extracted (relative to the median pixel height of the local region). The estimated boundaries of the streptavidin and Cas9/dCas9 were iteratively expanded or retracted around the original estimate until they expanded to a contiguous region greater than (μ_(d)+σ_(d)), where μ_(d) and σ_(d) are the mean and standard deviation of the height of the traced DNA beyond the estimated positions of bound proteins, and the estimate converges.

To account for any instrumental hysteresis which may distort the apparent length of DNA, the length of the DNA was normalized, and only DNA molecules originally measured to be 20% of their expected length (given the known number of base-pairs, 0.33 nm per base-pair) were used for further analysis (for the AAVS1 substrate—number traced: 804; nominal length: 1198 bp, mean length recorded: 1283 bp, std. dev: 154 bp; for the engineered substrate—number traced: 1520, nominal length: 986 bp, mean length recorded: 1071 bp, std. dev: 124 bp; for the ‘nonsense’ substrate—number traced: 616, nominal length: 1078 bp, mean length recorded: 1217 bp, std. dev: 135 bp). This step prevented us from improperly analyzing, e.g., two DNA molecules which appeared collinear, DNA which may have fragmented, or DNA which may have been cleaved by Cas9 and separated (which was rare, see main text).

The binding histograms of FIGS. 1C-1D, FIGS. 2C-2D, and FIG. 9 were generated by mapping the relative location of each bound protein to the bases overlapped (nearest-neighbor interpolation) by the protein and summing the total number of proteins bound to each site (if a single Cas9/dCas9 could be interpreted as being in contact with multiple (k) sites, each region of contact was weighed by 1/k in the binding histogram). Peaks in the binding histogram were fit to the empirical Gaussian exp(−((x−μ)/w)²), where μ is the mean peak position and w is the peak width parameter (w=√2σ, with σ the standard deviation), using MATLAB.

Determination of dCas9 Apparent Dissociation Constants. Apparent dissociation constants of dCas9 with different guide RNA variants were determined as previously described (Yang et al. (2005) Nucleic Acids Res., 33, 4322-4334). Briefly, at known solution concentrations of dCas9-guide RNA ([dCas9]₀) and DNA molecules ([DNA]₀), the respective numbers of ‘engineered’ DNA molecules were counted with and without proteins bound (fraction of DNA bound by proteins Θ_(dCas9)). After tracing DNA with bound proteins (see above) the average number of proteins bound per DNA molecule (n_(dCas9)) was determined. Overall dissociation constants are calculated as K_(d,DNA)=[DNA] [dCas9]/[DNA·dCas9]=(1−Θ_(dCas9)) ([dCas9]₀−n_(dCas9) [DNA]₀)/(Θ_(dcas9))

The protospacer-specific dissociation constants K_(d,protospacer) are calculated similarly using instead Θ_(dCas9,protospacer), the fractions of DNA with dCas9 bound within one peak width of the Gaussian fit in their respective binding histograms (i.e., see Table 1), as are the site-specific association constants K_(a,ss)=K_(d,ss) ⁻¹ using the fractions of each site on the DNA with a bound dCas9 Θ_(dCas9,ss).

Protein Alignment and Clustering. Images of Cas9 and dCas9 proteins which were isolated and appeared only to contact the DNA at a single location were extracted. These features were selected as those with features greater than (μ_(d)+2σ_(d)) which fit entirely within a 134 nm×134 nm bounding box, where d and ad are the mean and standard deviation of the DNA height to which the proteins are bound; this step essentially had the effect of removing most of the aggregated/densely packed Cas9/dCas9 from the set as well as those proteins from images with larger extrinsic noise. After four-fold nearest-neighbor interpolation, features of the protein with topographical height greater than (μ_(d)+σ_(d)) were each aligned by repeated translation, rotation, and reflection with respect to one another to minimize the mean-squared difference between their topographical heights. A distance matrix was composed of these minimized mean-square difference, then the proteins with standard sgRNA were clustered according to this criterion using the method of Rodriguez and Laio (27); proteins with the guide RNA variants were clustered according to the closest Cas9/dCas9 structure with the standard sgRNA. Ensemble average structures were extracted by performing a reference-free alignment across each member of individual clusters following the method of Penczek, Radermacher, and Frank (28). Properties of Cas9/dCas9 populations at each feature (such as protospacer sites) on the DNA were determined using proteins bound within one peak width of the Gaussian distributions fit to the binding histograms (i.e., see Table 1).

Kinetic Monte Carlo (KMC) of Guide RNA Strand Invasion and R-loop ‘Breathing’. Kinetic Monte Carlo (KMC) experiments to simulate strand invasion by the guide RNAs at protospacer sites were performed using a Gillespie-type (continuous time, discrete state) ((Gillespie (1976) Journal of computational physics, 22, 403-434) algorithm implemented in MATLAB. Strand invasion is modeled as a one-dimensional random walk in a position-dependent potential determined by the relative nearest-neighbor dependent DNA:DNA and RNA:DNA binding free energies. See, e.g., FIG. 4A. That is, the guide RNA is base-paired with the protospacer up to protospacer site m (1≥m≥20 for sgRNA and 1≥m≥18 for a truncated sgRNA (tru-gRNA)) and, to first-order, the forward rate (rate of additional guide RNA invasion) v_(f) is estimated using the symmetric approximation to be exp(−(ΔG° (m+1)_(RNA:DNA)−ΔG° (m+1)_(DNA:DNA))/2RT), where R is Boltzmann's constant, T is the temperature (here 37° C. to correspond with parameter set that was used), ΔG° (m+1)_(RNA:DNA) is free energy of the base-pairing between the RNA and protospacer at site m+1 and ΔG° (m+1)_(DNA:DNA) is the free energy of the base-pairing between the protospacer and its complementary DNA strand (the ½ corrective term is included to satisfy detailed balance). v_(f) at state m=20 or 18 for sgRNA or tru-gRNA was set to 0. The reverse rate (rate of re-hybridization between the protospacer and its complementary DNA strand) v_(r) is calculated similarly as proportional to exp(−(ΔG° (m)_(DNA:DNA)−ΔG° (m)_(RNA:DNA))/2RT); if state m=1, the simulation was halted (signifying guide RNA—protospacer dissociation). Starting at time t=0 (in arbitrary time units), for each iteration of the algorithm, the m-dependent rates are determined and two random numbers r₁ and r₂ are generated from a uniform distribution between 0 and 1. t is advanced by Δt=log(r₁)/(v_(f)+v_(r)). State m is increased to m+1 if r₂≥≥v_(f)/(v_(f)+v_(r)) or decreased to m−1 otherwise. For ‘equilibrium’ measurements of R-loop breathing, m was initiated at m=20 (or 18 in the case of tru-gRNA) and the algorithm iterated until t≥10,000. For measurements of ‘invasion’ kinetics' dynamics (such as in the presence of mismatched base-pairs), m was initiated at m=10 (up to t=1000).

Free energy parameters are derived from the literature from experiments at 1M NaCl at 37° C. Sequence-dependent DNA:DNA hybridization free energies ΔG° (x)_(DNA:DNA) were obtained from SantaLucia et al. (1996) Biochemistry, 35, 3555-3562; sequence-dependent RNA:DNA hybridization free energies ΔG° (x)_(RNA:DNA) were obtained from Sugimoto et al. (1995) Biochemistry, 34, 11211-11216; and ΔG° (x)_(RNA:DNA) values in cases of introduced point mismatches rG·dG, rC·dC, rA·dA, and rU·dT were obtained from Watkins et al. (2011) Nucleic acids research, 39, 1894-1902 (under slightly higher salt conditions). The sequence of the protospacer used is ‘ATCCTGTCCCTAGTGGCCCC’ (SEQ ID NO: 336), the AAVS1 target site as in the AFM experiments; the sequence of the protospacer complementary DNA is ‘GGGGCCACTAGGGACAGGAT’ (SEQ ID NO: 337), and the sequence of the guide RNA was either ‘GGGGCCACUAGGGACAGGAU’ (SEQ ID NO: 338) for sgRNA or ‘GGCCACUAGGGACAGGAU’ (SEQ ID NO: 339) for the truncated RNA.

Correlations between R-loop stability derived from KMC and experimental Cas9 cleavage rates. To analyze correlations between guide RNA—protospacer interactions and Cas9 cleavage rates in vivo, the sequences of guide RNAs and targeted DNA from Hsu et al. (2013) Nature biotechnology, 31, 827-832 and their experimentally determined maximum likelihood estimate (MILE) cutting frequencies by Cas9 were extracted. The sequences of guide RNAs and targeted DNA from Hsu et al. (2013) Nature Biotechnology, 31, 827-832 with single-nucleotide PAM-distal (≥10 bp away from the PAM site) mismatches of type rG·dG, rC·dC, rA·dA, and rU·dT and the experimentally determined maximum likelihood estimate (MLE) cutting frequencies by Cas9 at those sites were imported (n=136) into the KMC script. Simulations of strand invasion initiated at m=10 were repeated 1000 times for each sequence (up to t=100) to obtain the mean fraction of time m≥16 and correlated with the empirical cleavage rates. Significance was determined by bootstrapping the mean fraction of occupancy with the MILE cutting frequencies via permutation 100,000 times, then recalculating correlation coefficients and p-values. Guide RNA—protospacer binding free energies were estimated by summing over the nearest neighbor energies using the parameter sets listed above and corrected with a −3.1 kcal mol⁻¹ initiation factor.

dCas9-tru-gRNA and dCas9-hp-gRNA data for comparison with dCas9-sgRNA structural properties. When comparing height and volume measurements of the proteins across experiments, the AFM imaging conditions should remain mostly consistent so as not to introduce artifacts. This does not generally present an issue, for example, when comparing heights and volumes of dCas9 bound to different sites on the engineered DNA molecules, but presents a challenge when comparing the structural properties of dCas9/Cas9 when using different guide RNAs or DNA substrates. As a control, the heights and volumes of the streptavidin proteins used to label the ends of the traced DNA molecules were used, which should remain unchanged across all experimental conditions, for the different experiments. For experiments with sgRNAs, mean heights of the streptavidins differed by less than 0.1 nm (mean difference: 0.087 nm; standard deviation of differences: 0.052 nm) and their mean volumes (1098 nm³) differed by less than 15 nm³ (mean difference: 14.461 nm³; standard deviation of differences: 10.419 nm³). However, the mean heights and volumes between the experiments with tru-gRNA and the hp-gRNAs differed from those with sgRNAs by up to 0.14 nm and 225 nm³, respectively. To directly compare the results of these experiments, the heights of dCas9 with tru-gRNA and hp-gRNAs on engineered DNA were shifted by their difference in mean heights relative to those with sgRNAs and the volumes scaled by the percent difference of the mean volumes.

Example 2 Atomic Force Microscopy Captures Cas9/dCas9 Binding Specifically and Non-Specifically Along Engineered DNA Substrates with High Resolution

The analysis of crystallographic and biochemical experiments suggests that specificity in protospacer binding and cleavage is imparted first through the recognition of PAM sites by Cas9 itself, followed by strand invasion by the bound RNA complex and direct Watson-Crick base-pairing with the protospacer (FIG. 1A), although a complete mechanistic picture has yet to emerge. To directly probe the relative propensities to bind to protospacer and off-target sites with single-molecule resolution, 50 nM Cas9-sgRNA or dCas9-sgRNA complexes targeting the AAVS1 locus of human chromosome 19 were imaged by AFM in air after incubation with one of three DNA substrates (2.5 nM):

(i) a 1198 bp segment of the AAVS1 locus containing the complete target site following a PAM (here ‘TGG’) (FIG. 1C);

(ii) a 989 bp engineered DNA substrate containing a series of six complete, partial, or mismatched target sites each separated by approximately 150 bp (FIG. 1D). Mismatches at these sites could span both the ‘seed’ (PAM-proximal, approximately 12 bp) and ‘non-seed’ (PAM-distal) regions of the protospacer. The only PAM sites in this engineered substrate were at these explicitly designed locations; and

(iii) a 1078 bp ‘nonsense’ DNA with no homology (beyond 3 bp sequences) with the target sequence (FIGS. 9A-9C).

FIG. 1C shows that dCas9 and Cas9 exhibit nearly identical binding distributions on the AAVS1 substrate (n=404 and n=250, respectively). FIG. 1D shows that on the engineered substrate (n=536) dCas9 binds with the highest propensity to the complete protospacer with no mismatched (MM) sites (peak 1, later referred to as the full or ‘0 MM’ site) and also to sites with 5 or 10 mismatched bases distal to the PAM site (third and fourth feature from streptavidin label, referred to later as the ‘5 MM’ or ‘10 MM’ sites, respectively) albeit with the reduced affinity. Sites containing greater numbers of mismatches (second and fifth feature), or which possess two PAM-proximal mismatched nucleotides (sixth feature) are bound at significantly lower rates. (below) Distribution of PAM (‘TGG’) sites in each substrate.

Structurally, S. pyogenes Cas9 is a 160 kDa monomeric protein approximately 10 nm×10 nm×5 nm (from crystal structures), roughly divided into two lobe-like halves each containing a nuclease domain. Consistent with the x-ray structures, dCas9-sgRNA imaged via AFM appears as large ovular structures (FIGS. 10A-10C), after incubating Cas9 or dCas9 with DNA these structures bound along DNA were observed and assigned to be Cas9 or dCas9, respectively (FIG. 1B, FIGS. 10A-10C, and FIGS. 11A-11D). To unambiguously determine the sequence of the sites bound by Cas9 and dCas9, the biotinylated DNA molecules were labelled at one end with monovalent streptavidin tag prior to AFM imaging. DNA molecules that were observed with bound Cas9 or dCas9 proteins were selected for further analysis and traced with sub-pixel resolution according to a modified protocol adapted from that of Wiggins et al. (25), and the sites bound by Cas9/dCas9 were extracted (see Supplementary Methods for details).

This method proved remarkably robust (Table 1): on the DNA bound by Cas9 or dCas9, a distinct enrichment of proteins centred precisely at the location of protospacer sites with an adjoining PAM (within the expected 23 bp, FIG. 1C-D) is observed and manifest as sharp peaks. No such obvious peaks are observed in the DNA substrate containing no target sites (FIGS. 9A-9C). Standard deviation of the peak widths ranged from 36-60 bp, which is a significant improvement compared with binding experiments using single-molecule fluorescence that result in peak width standard deviations a of approximately 1000 bp). The mean apparent Cas9/dCas9 ‘footprint’ on DNA covering 78.1 bp±37.9 bp; this broadening of the apparent footprint over the ˜20 bp footprint of Cas9 on DNA determined by biochemical and crystallographic methods is a well-established result of imaging convolution with the width of the AFM tip. Previously, it had been observed in vitro that Cas9 remains bound to targeted DNA for extended periods (>10 min) after putative DNA cleavage as a single-turnover endonuclease, and could not be displaced from the cleaved strands without harsh chemical treatment. Most of the DNA molecules observed with bound Cas9 appeared as full-length AAVS1-derived substrates, with only a small (˜5%) percentage of substrates that have been both cleaved and separated. After these DNA molecules were traced, Cas9 was observed to bind to these ‘full-length’ substrates with nearly an identical distribution as was dCas9 (two-sided Komolgorov-Smirnov test, significance level 5%) (FIG. 1C).

TABLE 1 Peaks recorded in binding histograms of FIGS. 1C-D for Cas9/dCas9-sgRNA and FIG. 2C for dCas9 with sgRNAs possessing 2 nt truncation at 5′- end (tru- gRNA), based on empirical fit to Gaussian ∝ exp(-((x-μ)/w)²) Guide sgRNA^(a) tru-gRNA^(b) sgRNA RNA: Substrate: Engineered DNA: Engineered DNA: AAVs1-DNA: derivec Total DNA n = 536 n=257 n = n = molecules 404 250 traced:^(c) Location Full 10MM 5MM Full 10MM 5MM Full Full name: site sited site⁶ site site^(f) site® site site Cas9/dCas9 dCas9 dCas9 dCas9 dCas9 dCas9 dCas9 dCas9 Cas9 Location:^(h) 144- 452- 592-610 144- 452- 592- 316- 316- 167 465 167 465 610 339 339 Peak μ^(i) 151.3 467.6 600.6 159.0 462.9 592.0 327.7 315.0 (95% (151.1, (466.6, (599.5, (158.2, (462.1, (590.9, (327.3, (314.4, conf.): 151.6) 468.5) 601.7) 159.7) 463.6) 593.0) 328.2) 315.7) Peak 51.46 57.5 70.8 53.98 54.44 67.88 84.10 58.7 width^(i) w = (51.53, (55.84, (68.72, (52.2, (52.07, (64.27, (83.12 (56.8, √2σ 52.38) 59.16) 72.89) 55.76) 56.81) 71.49) 85.27) 60.63) (95% conf.): # dCas9^(k): 287 180.5 211.9 84.5 58.75 74.33 #/(2w) 1 0.5688 0.5399 1 0.6894 0.6994 (scaled to density at full site, 95% conf.): ^(a)Standard single-guide RNA (sgRNA) ^(b)Single-guide RNA with 2 nt truncated from 5′-end (tru-gRNA) ^(c)Numbers of DNA molecules observed with both monovalent streptavidin label and bound protein which were then traced (see Supporting Methods for details). ^(d)Target site with 10 PAM-distal mismatched nucleotides ^(e)Targeted site with 5 PAM-distal mismatched nucleotides ^(f)On the engineered DNA substrate, tru-gRNA is expected to interact with only the first 8 of the 10 PAM-distal mismatched nucleotides at the 10MM site. ^(g)On the engineered DNA substrate, tru-gRNA is expected to interact with only the first 3 of the 5 PAM-distal mismatched nucleotides at the 5MM site. ^(h)bp from streptavidin-labelled end (from PAM to end of site) ^(i)Peak maximum in binding histogram (from Gaussian fit) ^(j)Peak width is V2o, with o as the standard deviation ^(k)Number of dCas9 molecules observed within 1 peak width (√2σ) of binding site. If Cas9/dCas9 appeared to contact DNA at n sites, that molecule is weighted by 1/n. If molecules overlapped both 10 MM and 5 MM sites, # was weighted by an additional 1/2.

By examining the occupancies of dCas9 bound to different locations along the engineered substrate, the relative binding propensities of dCas9 to various mismatched and partial target sites could be determined (FIG. 1D, Table 1). The overall dissociation constant between dCas9 and the entire DNA substrate was estimated to be 2.70 nM (±1.58 nM, 95% confidence, Table 2). The dCas9 dissociation constant specifically at the site of the full (perfectly-matched) protospacer (within one peak width in the binding histogram) located substrate to be 44.67 nM (±1.04 nM, 95% confidence). Earlier electrophoretic mobility shift assays (EMSA) had estimated dCas9-sgRNA binding to protospacer sites on short DNA molecules (˜50 bp) to be between 0.5 nM and 2 nM. While the increase in dissociation constant at protospacer sites observed may be related the presence of multiple off-target sites on the engineered DNA substrate, it is typical that dissociation constants determined by AFM are nearly an order of magnitude higher than those determined by traditional assays (26). This difference is often attributed to nonspecific interactions of proteins to the blunt ends of the shorter DNA that are not accounted for in EMSA.

TABLE 2 Apparent dissociation constants for dCas9 with different guide RNA variants from the 989 bp ‘engineered’ DNA substrates (e.g., FIGS. 1D, 2C, and 2D) that contain a series of fully- and partially-complementary protospacer sites Overall dissociation Protospacer-specific constant between dissociation constant dCas9 and the for dCas9 and engineered the full target on the Guide RNA DNA substrate engineered substrate variant (±95% confidence) (±95% confidence) sgRNA^(a) 2.70 nM(± 1.58 nM) 44.67 nM (±1.04 nM) tru-gRNA^(b) 17.89 nM(± 0.45 nM) 136.4 nM (±2.30 nM) hp6-gRNA^(c) 16.61 nM (± 0.40 nM) 164.4 nM (±13.63 nM) hp10-gRNA^(d) 35.84 nM (± 0.63 nM) 164.8 nM (±15.60 nM) ^(a)Full-length single-guide RNA (sgRNA) ^(b)Truncated sgRNA (first two nt at 5′- truncated) ^(c)sgRNA with additional 5′- hairpin which overlaps six PAM-distal targeting nts (see text) ^(d)sgRNA with additional 5′- hairpin which overlaps ten PAM-distal targeting nts (see text)

On the engineered substrate, dCas9 is relatively tolerant to distal mismatches (exhibiting 50-60% binding propensity relative to complete target site, FIG. 1D and Table 1), and has the same apparent affinity (within confidence) toward target sites containing 5 and 10 distal mismatches (MMs). However, binding to protospacer sites containing only two PAM-adjacent mismatches occurred with similar propensity as to sites with 15 or even 20 (PAM site alone) distal mismatches (approximately 5-10% binding propensity relative to perfect target, approximately that of the background binding signal), a finding consistent with previous biochemical studies. While there are no PAM sites on the engineered substrate except adjacent to the protospacer sites, on the AAVS1-derived substrate there is a distinct ‘shoulder peak’ of enhanced Cas9 and dCas9 binding near the AAVS1 target that is particularly enriched in PAM sites. On the ‘nonsense’ substrate and the segments of the AAVS1-derived substrate away from target sites, subtle enrichments of dCas9 closely mirrored the distribution of PAM sites (two-sided Komolgorov-Smirnov test, significance level 5%) and dCas9 distribution on the ‘nonsense’ substrate more closely reflected the experimental PAM distribution than it did to 71.20% of 100,000 randomly generated sequences with the same dA, dT, dC, and dG distributions (FIGS. 9A-9C). As dCas9 binding along the ‘nonsense’ substrate (with 879 PAM sites in 1079 bp) corresponds so well with PAM site distribution, this was interpreted as a measurement of real dCas9-PAM interactions. The mean single-site dissociation constant for dCas9 binding along the ‘nonspecific’ substrate was estimated to be approximately 867 nM (standard deviation+209 nM). This can be understood as an estimate of the dCas9 binding dissociation constant on DNA with no protospacer homology.

Example 3 sgRNAs with a Two Nucleotide Truncation at their 5′-Ends (Tru-gRNAs) do not Increase Binding Specificity of dCas9 In Vitro

Cas9 was found to still exhibit cleavage activity even if up to four nucleotides of the guide (protospacer-targeting) segment of the sgRNA or crRNA were truncated from their 5′-ends and Fu et al. (21) recently showed that use of sgRNAs with these 5′-truncations (optimally by 2-3 nucleotides) can actually result in orders-of-magnitude increase in Cas9 cleavage fidelity in vivo. It was suggested that the increased sensitivity to mismatched sites (MM) using these truncated sgRNAs (termed ‘tru-gRNAs’, FIG. 2A) was a result of its reduced binding energy between the guide RNA and protospacer sites. This implies that the binding energy imparted by the additional 5′-nucleotides on the sgRNA could compensate for any mismatched nucleotides and stabilize the Cas9 at incorrect sites, while the tru-gRNAs would be relatively less stable on the DNA if there are mismatches.

As a test of this proposed mechanism, dCas9 was imaged with a tru-gRNA with a two nucleotide 5′-truncation relative to the sgRNA used previously. The dCas9-tru-gRNA complexes were incubated with the engineered substrates that contained a series of full and partial protospacer sites. Again a distinct peak was found precisely at the full protospacer site (FIG. 2C and Table 1), although the apparent association constant relative to dCas9 with a full sgRNA at this site decreases considerably (i.e., dissociation constant increases, see Table 2). However, relative to binding at full protospacer sites, off-target binding by dCas9 with the tru-gRNA at the protospacer sites with PAM-distal mismatches actually increases when compared to dCas9 with sgRNAs (FIG. 2C and Table 1). Similar to dCas9 with sgRNA, dCas9 with tru-gRNA binds to protospacers with either 10 or 5 PAM-distal mismatched sites with approximately equal propensities (note that the tru-gRNA is only expected to interact with the first 8 and 3 mismatches at those sites, respectively). These results suggest that increased cleavage fidelity using tru-gRNAs is not necessarily imparted by a relative reduction of binding propensity at off-target sites or a reduction in relative stability in the presence of mismatches. Rather, while there may be some ‘threshold’ effects where reduction of the association constant below ˜4-5×10⁶ M effectively abolishes cleavage activity in vivo, these and additional results presented below suggest that the increased specificity exhibited by the tru-gRNAs may be influenced by discrimination in the cleavage mechanism itself. Furthermore, these findings would suggest that while tru-gRNAs can improve specificity in cleavage of active Cas9, they may not improve specificity in their binding activity for applications involving dCas9 (or chimeric derivatives) in vivo.

Additionally, previous reports have shown that tru-gRNAs, which have 5′-truncations (optimally by 2-3 nucleotides), in their protospacer-targeting segment can result in orders-of-magnitude increase in Cas9 cleavage fidelity in vivo (FIG. 2A), the results shown in the Examples indicate that the truncated gRNAs do not improve specificity in dCas9 binding (FIG. 2C). FIG. 2C shows the binding affinity of dCas9 with a standard gRNA (dashed line) compared with the binding affinity of a dCas9 with a tru-gRNA (trugRNA, purple line) on a DNA molecule which contains a full protospacer (site i) as well as protospacer sites with 5 and 10 PAM-distal mismatches (sites ii and iii, respectively). FIG. 2C shows the standard guide RNAs retain significant ability to bind to these off-target sites (containing mismatches), and that trugRNAs exhibit no relative enhancement in binding specificity at sites which contained mismatches in the 5 10 nucleotides at the PAM distal end of the protospacer. The binding distribution of dCas9 with tru-gRNAs exhibits distinct peaks in its affinity exactly at the protospacer sites with 10 PAM-distal mismatches and 5 PAM-distal mismatches, demonstrating that it does not have increased binding specificity relative to full sgRNAs (see Table 1). The ‘peaks’ in the binding histogram are indicative of specific, stable binding at these off-target sites. In fact, binding at the off-target sites by dCas9-trugRNAs actually increases relative to binding to the protospacer compared to the standard guide RNA. This promiscuous binding may limit their utility for dCas9 and chimeric dCas9 derivatives. It may also reflect the off-target cleavage reported for this system which, while improved relative to the standard guide RNAs, was still significant at some off-target sites. For comparison, we found no specific binding of the hpgRNAs at these sites with mismatches (FIG. 2D). hpgRNAs bound at these sites with approximately the same affinity as they do nonspecifically to DNA with no homology to the protospacer, with a ˜22% decrease in the maximum observed off-target binding affinities relative to the truncated gRNAs. Additionally, based on the narrow geometry of the Cas9 DNA-binding channel, we expect that the presence of an unopened hairpin at mismatched protospacers may inhibit the conformational change in Cas9 necessary to perform cleavage (FIG. 1 ).

Significant efforts have been made to characterize this off-target activity—and to improve specificity of Cas9/dCas9 through intelligent selection of protospacer target sequences; optimization of sgRNA structure, for example, by truncation of first two 5′-nucleotides in the sgRNA; and use of ‘dual-nicking’ Cas9 enzymes—but a clear understanding of the precise mechanism of RNA-guided cleavage as it relates to the structural biology of Cas9 will be essential to developing Cas9 derivatives and guide RNAs with increased fidelity for their emerging applications in medicine and biology.

Pursuant to this goal, here we use atomic force microscopy (AFM) to resolve individual S. pyogenes Cas9 and dCas9 proteins as they bind to targets along engineered DNA substrates after incubation with different sgRNA variants. This technique allows us to directly resolve both the binding site and structure of individual Cas9/dCas9 proteins simultaneously, providing a wealth of mechanistic information regarding Cas9/dCas9 specificity with single-molecule resolution. Consistent with traditional biochemical studies, we find that significant binding by Cas9/dCas9 with sgRNAs occurs at sites containing up to 10 mismatched base-pairs in the target sequence. However, while use of guide RNAs with two nucleotides truncated from their 5′-end (tru-gRNA) had previously shown to result in up to 5000-fold decrease in off-target mutagenesis by Cas9 in vivo, we find similar specificities in vitro for dCas9 with tru-gRNA binding to mismatched targets as with standard sgRNA. The addition of a hairpin to the 5′-end of the sgRNA which partially overlaps the target-binding region of the guide RNA is found to increase dCas9 specificity at the cost of overall decreased binding propensity to DNA. Our results indicate that overall stability of guide RNA-DNA binding does not necessarily govern specificity in Cas9 cleavage when mismatches are located more than 10 bp away from the PAM.

Example 4 Guide RNAs with 5′-Hairpins Complementary to ‘PAM-Distal’-Targeting Segments (Hp-gRNAs) Modulate the Absolute Binding Propensity and Profile of dCas9s Bound to DNA with Mismatched Protospacers In Vitro

dCas9 specificity may be increased by extending the 5′-end of the sgRNA such that it formed a hairpin structure which overlapped the ‘PAM-distal’-targeting (or ‘non-seed’) segment of the sgRNA (FIG. 2B). After a PAM site is bound and strand invasion of the DNA by the guide RNA has initiated, the hairpin is opened upon binding to a full protospacer and full strand invasion can occur. If there are PAM-distal mismatches at the target site, then it is more energetically favourable for the hairpin to remain closed and strand invasion is hindered. Similar topologies have been used recently for ‘dynamic DNA circuits’ which are driven by strand invasion. In those systems, the hairpins serve as kinetic barriers to invasion, with oligonucleotide invasion rates slowed several orders of magnitude in cases of attempted invasion by targets with mismatches. The hairpins here may be displaced during invasion of the full target sites, but inhibit invasion if there were mismatches between the target and the non-seed targeting region of the guide RNA (FIG. 2B). In those cases, it is more energetically favourable for the hairpins to remain closed. While previous efforts which had added 5′-extensions to sgRNAs in order to complement additional nucleotides beyond the protospacer, these guide RNAs did not show increased Cas9 cleavage specificity in vivo. Rather, they were digested back approximately to their standard length in living cells. Based on the size and structure of the hairpin, the hairpin may be accommodated within the DNA-binding channel of Cas9/dCas9 molecule and protected from degradation.

sgRNAs were generated with 5′-hairpins (hp-gRNAs) which overlapped the nucleotides complementary to the last six (hp6-gRNA) or ten (hp10-gRNA) PAM-distal sites of the protospacer. By mapping the observed binding locations of dCas9-hp-gRNAs on the engineered DNA substrate (FIG. 2D), sharp peaks were observed precisely at the protospacer site (PAM and protospacer located at sites 144-167, with binding peak at site 154.0 (95% confidence: 153.3-154.8) for dCas9-hp6-gRNA and at 158.3 (95% confidence: 157.6-158.9) for dCas9-hp10-gRNA). The specific peaks at the sites with 5 and 10 distal mismatches are significantly flattened, with dCas9 and hp10-gRNA exhibiting substantially decreased affinity for off-target sites (22% drop relative to dCas9 with tru-gRNA). The peaks in affinity at the full protospacer sites imply that the hairpins indeed open upon full invasion. n=243 for hp6-gRNA and n=212 for hp10-gRNA. dCas9 with hp-gRNAs show a similar drop in affinity for the target site as with tru-gRNAs, however, in contrast to dCas9 with tru-gRNAs, dCas9 with hp-RNAs do not present any sharp binding peaks at off-target sites which would otherwise indicate strong, specific binding. With hp6-gRNA, there was an enrichment of binding around the sites of protospacers with 5 or 10 mismatched PAM-distal sites. Because they lack the sharp binding peaks observed with sgRNA and tru-gRNA, these enrichments are not likely indicative of specific binding, but rather may indicate that the dCas9 had dissociated from these sites upon adsorption to the surface. This would indicate very weak binding at those off-target sites in the case of hp6-gRNA.

In the case of hp10-gRNA, binding to these mismatched sites is approximately at the level of the non-specific binding elsewhere on the substrate, representing a 22% decrease in the maximum observed off-target binding affinity relative to the tru-gRNAs (decrease in the maximum observed association constant from to 3.18×10⁶ M to 2.48×10⁶ M, FIG. 2D). This increase in specificity of hp10-gRNA is also reflected by a similar binding dissociation constant as hp6-gRNA to the protospacer sites but a significant increase in the overall dissociation constant to the entire (specific+non-specific) engineered substrate relative (Table 2).

The distinct enrichment precisely at the complete protospacer sites suggests that upon invasion of full protospacer sites the hairpins in the hp-gRNAs are in fact opening, as the nucleotides which bind the PAM-distal sites of the protospacer would otherwise be trapped within the hairpin. A likely mechanism for the improvement of binding specificity is that, when unopened at protospacer sites with PAM-distal mismatches, the presence of the hairpin promotes melting of the guide RNA from these off-target sites. The results suggest that the hp-gRNAs can be used to tune Cas9/dCas9 binding affinities and specificity, and further manipulation of hairpin length, loop length, and loop composition may allow for more fine control of these properties.

Example 5 Cas9 and dCas9 Undergo a Progressive Structural Transition as they Bind to DNA Sites that Increasingly Match the Targeted Protospacer Sequence

It was observed using negative-stain transmission electron microscopy (TEM) that, upon binding sgRNA, the structure of dCas9 compacts and rotates to open a putative DNA-binding channel between its two lobes. After binding to DNA containing the PAM and protospacer sequence, dCas9 undergoes a second structural reorientation to an expanded conformation. The role of this second transition was suggested to be related to strand invasion by sgRNA or to align the two major Cas9 nuclease sites with the two separated DNA strands. However, these studies were performed only in the presence or absence of DNA containing fully-matched protospacer sequences, and examining the transition between these conformations at partially matched protospacer sites can provide insights into the mechanism of off-target binding and cleavage. Therefore, in addition to determining relative binding propensities, AFM imaging was used to capture these putative conformational transitions by Cas9 and dCas9 as they bind to DNA at sites of various complementarity to the protospacer. We extracted the volumes and maximum topographical heights of Cas9 and dCas9 proteins with sgRNAs which appeared isolated on the DNA (n=839) and mapped these values to their respective binding sites on DNA (FIG. 3 , FIGS. 11A-11D, and FIGS. 12A-12B). The binding site distribution is nearly identical to the distribution of the full data set, indicating that this selection was unbiased and representative. The recorded image of each of these proteins was extracted (FIGS. 11C-11D) and aligned pair-wise by iterative rotation, reflection, and translation. The protein structures was clustered according to their pair-wise mean-squared topographical difference (FIGS. 12A-12B and Table 3). A pronounced advantage of this technique is that it naturally clusters any monovalent streptavidin or any aggregated Cas9/dCas9 proteins that co-localize on the surface with the DNA separately from those assigned to be individual Cas9/dCas9 molecules, allowing for an unbiased analysis of the structural properties of these proteins on DNA. Analysis of the distribution of binding sites by either the putative streptavidin molecules or aggregated proteins reveals that they are both rare and uniformly distributed along the DNA and hence did not interfere with analysis of the binding site distributions (FIGS. 12A-12B).

At sites containing no homology to targets, such as on the ‘nonsense’ DNA substrate, dCas9 molecules with sgRNAs were predominately smaller and egg-shaped (FIG. 3C(iii), and Table 3). But as dCas9 proteins bind to increasingly complementary target sequences (FIG. 3 (α-δ)), their height and volume significantly increase (FIGS. 3D and 12A-12B, Table 2) relative to non-specific binding, reaching a maximum size at the protospacer sequence. This increase is likewise accompanied by a shift in the population of dCas9 (FIGS. 3A, and 12A-12B, Table 2) from structures clustering with the flatter and egg-shaped conformations (FIGS. 3C(ii) and 3C(iii), blue and green) to those which increasingly cluster with slightly rounder structures possessing a large, central bulge (FIG. 3C(i), yellow). This latter observed conformation is likely the expanded conformation previously observed via TEM and recently by size exclusion chromatography, and is presumably the active state where the nuclease domains of Cas9 are positioned properly around the DNA such that cleavage could occur most efficiently.

Catalytically active Cas9 undergoes a significant increase in size as it binds to the protospacer sequence as well (FIG. 3(F)); however there is a small, but statistically significant, decrease in size relative to dCas9, and the conformation of Cas9 at full protospacer sites tends to cluster with the flatter (green) structures. As we do not concurrently monitor whether the DNA has been cleaved at the time of imaging, it is unclear if this represents another conformational change after DNA cleavage or is a result of the mutational differences between Cas9 and dCas9; however as binding and strand invasion have been previously determined to be the rate-limiting steps it is likely that the DNA within the Cas9 is cleaved during these measurements.

TABLE 3 Properties of dCas9/Cas9 with different guide RNA variants at fully-, and partially-, and non-complementary protospacer sites Mean Volume Guide (nm^(3 ×) 10⁴) ± Mean Height Site DNA RNA n^(a) SEM^(b) (nm) ± SEM Protospacer Engineered + sgRNA 201 0.6226 ± 0.016 1.932 ± 0.041 (dCas9) AAVsI^(c) Y: 41% (±6.8%) G: 22% (±5.8%) B: 21% (±5.6%) Protospacer AAVsI sgRNA  65 0.5784 ± 0.035 1.753 ± 0.076 (Cas9) Y: 17% (±9.1%) G: 32% (±11.4%) B: 26% (±10.7%) 10MM Engineered sgRNA  76 0.5510 ± 0.011 1.601 ± 0.026 (dCas9) Y: 25% (±8.9%) G: 31% (±9.7%) B: 34% (±9.9%)  5MM Engineered sgRNA  85 0.6055 ± 0.024 1.790 ± 0.049 (dCas9) Y: 34% (±8.8%) G: 34% (±8.8%) B: 25% (±8.1%) Non- AAVsI + sgRNA 274 0.4780 ± 0.015 1.553 ± 0.034 specific Nonsense^(c) Y: 21% (±4.8%) (Cas9 + G: 17% (±4.5%) dCas9) B: 39% (±5.8%) Protospacer Engineered tru-  47 0.5421 ± 0.041 1.761 ± 0.079 (dCas9) ^(d) gRNA^(g) Y: 26% (±12.5%) G: 17% (±10.7%) B: 34% (±13.6%) (10MM) Engineered tru-  32 0.5123 ± 0.049 1.665 ± 0.099 (dCas9)^(d,e) gRNA Y: 13% (±11.5%) G: 38% (±16.7%) B: 19% (±13.5%) (5MM Engineered tru-  34 0.5346 ± 0.048 1.705 ± 0.084 (dCas9)^(d,f) gRNA Y: 18% (±12.8%) G: 29% (±15.3%) B: 24% (±14.2%) Non- Engineered tru-  72 0.4554 ± 0.035 1.532 ± 0.059 specific gRNA Y: 14% (±8.0%) (dCas9) G: 17% (±8.6%) B: 29% (±10.5%) Protospacer Engineered hp6-  47 0.5940 ± 0.043 1.860 ± 0.109 (dCas9) gRNA^(g) Y: 26% (±12.5%) G: 17% (±10.7%) B: 34% (±13.6%) Non- Engineered hp6-  32 0.4656 ± 0.024 1.572 ± 0.047 specific gRNA Y: 13% (±11.5%) (dCas9) G: 38% (±16.7%) B: 19% (±13.5%) Protospacer Engineered hp10-  47 0.6304 ± 0.038 1.837 ± 0.076 (dCas9) gRNA^(g) Y: 26% (±12.5%) G: 17% (±10.7%) B: 34% (±13.6%) Nonspecific Engineered hp10-  32 0.5181 ± 0.027 1.644 ± 0.050 (dCas9) gRNA Y: 13% (±11.5%) G: 38% (±16.7%) B: 19% (±13.5%) ^(a)Total molecules observed within two standard deviations of those sites. Below: fraction of population in the main three structural clusters (±95% binomial confidence) coloured as in FIG. 2 in main text (Y = yellow cluster, G = green cluster, B = light blue cluster). Full distribution of properties by cluster in FIGS. 12A-12B. ^(b)Standard error of the mean ^(c)Standard error of the mean ^(d)Rejected null hypothesis of height-volume distributions’ being different (p > 0.05; Hotelling’s T² test) ^(e)On the engineered DNA substrate, tru-gRNA is expected to interact with only the first 8 of the 10 PAM-distal mismatched nucleotides at the 10MM site (labelled ‘8MM’ in FIG. 3D). ^(f)On the engineered DNA substrate, tru-gRNA is expected to interact with only the first 3 of the 5 PAM-distal mismatched nucleotides at the 5MM site (labelled ‘3MM’ in FIG. 3D). ^(g)See Supplementary Comment 1 in Supporting Information regarding correction of the heights and volumes of proteins with tru-gRNA and hp-gRNAs so they could be compared to those with sgRNA.

Example 6 Interactions Between the Guide RNA and the Target DNA at or Near the 16^(th) Protospacer Site Stabilize the Cas9/dCas9 Conformational Change

AFM imaging directly reveals that although dCas9/Cas9 retains a significant propensity to bind protospacer sites with up to ten distal mismatches, binding to DNA sites that are increasingly complementary to the protospacer drives an increasing shift in the population of dCas9/Cas9 proteins toward what appear to be the active conformation. Notably, we see similar shift in structure between off-target sites and perfectly-matched sites for dCas9 with hp-gRNAs as well (Table 2 and FIG. 13 ). The presence of complementary PAM-distal sequences is known to be associated with increased stability of Cas9 on DNA. It was also recently found that Cas9 binding to single-stranded DNA with increasing PAM-distal complementarity to the protospacer (from 10 to 20 sites) resulted in an increased change of protein size. This was also then associated with a transition of Cas9 activity from nicking behaviour to full cleavage. Here, we directly can determine the volumes of Cas9/dCas9 bound onto double-stranded DNA sites. An analysis of the structural properties of individual Cas9/dCas9 proteins on double-stranded DNA reveals a steady conformational transition with increasingly matched target sequences that is consistent with a ‘conformational gating’ mechanism, where sgRNA base-pairing with these distal sites also stabilizes the active conformation so that efficient cleavage may occur, whereas binding to sites with numerous distal mismatches shifts the equilibrium away from the active structure (i.e., see FIG. 4D).

Along these lines, we see this effect is dramatically muted for dCas9 with the tru-gRNA (FIG. 3D and Table 3), with a smaller shifts between the structural populations within which the proteins cluster (FIG. 13 ). Additionally, while we see a statistical difference between the height-volume properties of dCas9-tru-gRNAs that are non-specifically bound and those bound at full or partial protospacer sites (p<0.05; Hotelling's T² test), at sites that increasingly match the protospacer (10 MM, 5 MM, and full protospacer sites) their structural properties are not statistically differentiable (FIG. 3D and Table 3). It was recently postulated that while invasion of the first 10 bp of the protospacer initiates a conformational change in Cas9, full invasion of the protospacer by the guide RNA helps to drive a further shift to the complete active state. We therefore hypothesized the observed depression of the conformational change at increasingly matched protospacer sites for dCas9 with tru-gRNAs (relative to those with sgRNAs) was a result of the decreased stability of these guide RNAs at PAM-distal sites.

To investigate the relative stabilities of sgRNAs and tru-gRNAs at these sites, we performed a kinetic Monte Carlo (KMC) study of the dynamic structure of the R-loop—that is, the structure formed by the invading guide RNA bound to a segment of contiguous DNA, exposing a single-stranded loop of the that segment's complementary DNA (FIG. 4A)—during and after strand invasion. See Supplementary Methods for more detail. Briefly, using a Gillespie-type algorithm, we modelled the strand invasion of the guide RNA bound up to protospacer site m as a sequential, nucleotide-by-nucleotide competition between invasion (breaking of base-pairing between the protospacer and its complementary DNA strand, then replacement with a protospacer-guide RNA base-pair) and re-annealing (the reverse), with sequence-dependent rates of invasion and re-annealing v_(f) and v_(r), respectively (FIG. 4A). To first-order, we approximate the transition rate from state m to m+1, v_(f), to be proportional to exp(−(ΔG° (m+1)_(RNA:DNA)−ΔG°(m+1)_(DNA:DNA))/2RT), where ΔG° (m+1)_(RNA:DNA) is free energy of the base-pairing between the RNA and protospacer at site m+1 and ΔG° (m+1)_(DNA:DNA) is the free energy of the base-pairing between the protospacer and its complementary DNA strand at m+1 (R is the ideal gas constant, T is the temperature, and the ½ term is added to satisfy detailed balance). v_(r) is estimated similarly as proportional to exp(−(ΔG° (m)_(DNA:DNA)−ΔG° (m)_(RNA:DNA))/2RT). Transition rates of this type have been previously used for computational studies of nucleotide base-pairing and stability, and here they allowed us to capture the general dynamics of the R-loop in a sequence-dependent manner.

In general, RNA:DNA base-pairs are energetically stronger than DNA:DNA base-pairs, and at equilibrium we see from the KMC trajectories that the guide RNAs are stably bound to the protospacer, as expected (FIG. 4C). However, while sgRNA is quite stable and remains nearly totally invaded—during 95% of simulated time course, the strand remains invaded up to the 19^(th) protospacer site (FIG. 4B)—tru-gRNA exhibits significant fluctuations of protospacer re-annealing at PAM-distal sites (FIGS. 4B and 4C). Because the only difference between the dCas9-sgRNA and dCas9-tru-gRNA is a simple truncation of two 5′-nucleotides from the guide RNA, and because we see an inhibition of the conformational change by dCas9-sgRNA at sites containing 5 PAM-distal mismatches, these results suggest that the conformational change to a fully active state is stabilized by interactions between the guide RNA and protospacer near the 16^(th) site of the protospacer, which is disrupted by the instability of the tru-gRNA in that region. In fact, the KMC experiments show that the mean lifetime between full invasion and re-annealing of the DNA back to the 16^(th) site is decreased by two orders of magnitude when replacing the sgRNA with the tru-gRNA (FIG. 4C inset). This result is consistent with the earlier finding that while Cas9 activity with tru-gRNA variants with 2 or 3 nucleotide (nt) truncations was modulated depending on sequence context, and that cleavage in all tested cases was dramatically reduced by ˜90%-100% by 4 nt truncations and abolished after a 5 nt truncation. The conformational change to the protein activate state is stabilized by these interactions at or near the 16^(th) site of the protospacer. This finding is supported by gRNA stability at the 14^(th)-17^(th) protospacer positions, which was estimated from additional KMC experiments described below and correlated with experimental off-target cleavage in vivo (see below) while stability of the guide RNA at protospacer sites 18-20 was not.

Example 7 Fluctuations of the Guide RNA-Protospacer R-Loop Suggest a Mechanism of Mismatch Tolerance by Cas9/dCas9 and of Increased Specificity in Cleavage by Tru-gRNAs

To investigate mechanisms by which Cas9 or dCas9 can tolerate or become sensitized to mismatches in protospacers, we performed a series of KMC experiments using the AAVS1 protospacer site where one or two PAM-distal (≥10 bp away from the PAM) mismatches were introduced (FIG. 5 ). Cas9 is generally more tolerant of PAM-distal mismatches than PAM-proximal mismatches. However, Hsu et al. (2013) Nature Biotechnology, 31, 827-832 identified significant and varying differences in estimated Cas9 cleavage rates at protospacers containing PAM-distal mismatches depending on sequence context, type of mismatch, and site of the mismatch. Based on our AFM and earlier KMC experiments, we hypothesized the differences in cleavage rates may similarly be a result of different stabilities of the guide RNA near the 16^(th) site of the protospacer. For these simulations, we only examined sequences with protospacers-guide RNA pairs which would result in isolated rG·dG, rC·dC, rA·dA, and rU·dT mismatches, for which the sequence context-dependent thermodynamic data is the most complete and suitable for our KMC model. The effects of these mismatched base-pairs are not expected to lower the overall binding energy between sgRNA and the protospacer dramatically (Table 4); for example, single rG·dG, rC·dC, rA·dA, and rU·dT mismatches lower RNA:DNA melting temperatures on average by 1.7° C. Rather, their effect is expected to be kinetic rather than thermodynamic in nature by hindering strand displacement at the mismatch. Hence we initiated the kinetic Monte Carlo experiments as proceeding from the 10^(th) protospacer site (initial R-loop length m=10), such as would be occurring during strand invasion.

TABLE 4 Sequences and Maximum Likelihood Estimate (MLE) Cutting Frequencies from Hsu et al. (2013) Nature Biotechnology, 31, 827-832 used for correlation analysis (mismatch site in target sequence bold). MLE Protospacer- Cutting Estimated SEQ targeting SEQ Frequency ΔG°₃₇ ID region of ID (Hsu et al. (kcal/m Target sequence NO Guide RNA NO (2013)) ol) TTCTTCTTCTGCTCGG 13 GUGUCCGAGCAGAAGA 149 0.10384 −32.16 ACTC AGAA TTCTTCTTCTGCTCGG 14 GACUCCGAGCAGAAGA 150 0.12609 −31.4 ACTC AGAA TTCTTCTTCTGCTCGG 15 GAGACCGAGCAGAAGA 151 0.13145 −32.69 ACTC AGAA TTCTTCTTCTGCTCGG 16 GAGUGCGAGCAGAAGA 152 0.097464 −32.33 ACTC AGAA TTCTTCTTCTGCTCGG 17 GAGUCGGAGCAGAAGA 153 0.12704 −33.43 ACTC AGAA TTCTTCTTCTGCTCGG 18 GAGUCCCAGCAGAAGA 154 0.079556 −31.37 ACTC AGAA TTCTTCTTCTGCTCGG 19 GAGUCCGUGCAGAAGA 155 0.11197 −32.36 ACTC AGAA TTCTTCTTCTGCTCGG 20 GAGUCCGACCAGAAGA 156 0.04788 −31.9 ACTC AGAA TTCTTCTTCTGCTCGG 21 GAGUCCGAGGAGAAGA 157 0.085461 −32.83 ACTC AGAA TTCTTCTTCTGCTCGG 22 GAGUCCGAGCUGAAGA 158 0.074938 −32.22 ACTC AGAA TTCTTCTTCTGCTCGG 23 GUGUCCGAGCAGAAGA 159 0.15588 −32.16 ACTC AGAA TTCTTCTTCTGCTCGG 24 GACUCCGAGCAGAAGA 160 0.11015 −31.4 ACTC AGAA TTCTTCTTCTGCTCGG 25 GAGACCGAGCAGAAGA 161 0.11435 −32.69 ACTC AGAA TTCTTCTTCTGCTCGG 26 GAGUGCGAGCAGAAGA 162 0.15072 −32.33 ACTC AGAA TTCTTCTTCTGCTCGG 27 GAGUCGGAGCAGAAGA 163 0.11567 −33.43 ACTC AGAA TTCTTCTTCTGCTCGG 28 GAGUCCCAGCAGAAGA 164 0.070181 −31.37 ACTC AGAA TTCTTCTTCTGCTCGG 29 GAGUCCGUGCAGAAGA 165 0.10538 −32.36 ACTC AGAA TTCTTCTTCTGCTCGG 30 GAGUCCGACCAGAAGA 166 0.064145 −31.9 ACTC AGAA TTCTTCTTCTGCTCGG 31 GAGUCCGAGGAGAAGA 167 0.085148 −32.83 ACTC AGAA TTCTTCTTCTGCTCGG 32 GAGUCCGAGCUGAAGA 168 0.064903 −32.22 ACTC AGAA CCCTAGTCATTGGAGG 33 GACACCUCCAAUGACUA 169 0.062949 −32.19 TGAC GGG CCCTAGTCATTGGAGG 34 GUGACCUCCAAUGACUA 170 0.063313 −31.73 TGAC GGG CCCTAGTCATTGGAGG 35 GUCUCCUCCAAUGACUA 171 0.068655 −31.72 TGAC GGG CCCTAGTCATTGGAGG 36 GUCAGCUCCAAUGACUA 172 0.073003 −32 TGAC GGG CCCTAGTCATTGGAGG 37 GUCACGUCCAAUGACUA 173 0.037401 −32.63 TGAC GGG CCCTAGTCATTGGAGG 38 GUCACCACCAAUGACUA 174 0.038197 −32.11 TGAC GGG CCCTAGTCATTGGAGG 39 GUCACCUGCAAUGACUA 175 0.041758 −31.63 TGAC GGG CCCTAGTCATTGGAGG 40 GUCACCUCGAAUGACUA 176 0.067751 −32.23 TGAC GGG CCCTAGTCATTGGAGG 41 GUCACCUCCUAUGACUA 177 0.031653 −31.62 TGAC GGG CCCTAGTCATTGGAGG 42 GUCACCUCCAUUGACUA 178 0.027161 −31.77 TGAC GGG ATGGGGAGGACATCG 43 GUCAUCGAUGUCCUCCC 179 0.027124 −31.26 ATGTC CAU ATGGGGAGGACATCG 44 GAGAUCGAUGUCCUCCC 180 0.022366 −31.7 ATGTC CAU ATGGGGAGGACATCG 45 GACUUCGAUGUCCUCCC 181 0.01127 −30.92 ATGTC CAU ATGGGGAGGACATCG 46 GACAACGAUGUCCUCCC 182 0.011836 −31.44 ATGTC CAU ATGGGGAGGACATCG 47 GACAUGGAUGUCCUCCC 183 0.009146 −31.83 ATGTC CAU ATGGGGAGGACATCG 48 GACAUCCAUGUCCUCCC 184 0.006333 −30.27 ATGTC CAU ATGGGGAGGACATCG 49 GACAUCGUUGUCCUCCC 185 0.006232 −31.06 ATGTC CAU ATGGGGAGGACATCG 50 GACAUCGAAGUCCUCCC 186 0.007085 −31.64 ATGTC CAU ATGGGGAGGACATCG 51 GACAUCGAUCUCCUCCC 187 0.001545 −30.32 ATGTC CAU ATGGGGAGGACATCG 52 GACAUCGAUGACCUCCC 188 0.00025 −31.59 ATGTC CAU ATCACATCAACCGGTG 53 GGGCCACCGGUUGAUG 189 0.15963 −35.23 GCGC UGAU ATCACATCAACCGGTG 54 GCCCCACCGGUUGAUGU 190 0.14121 −32.17 GCGC GAU ATCACATCAACCGGTG 55 GCGGCACCGGUUGAUG 191 0.18743 −33.43 GCGC UGAU ATCACATCAACCGGT 56 GCGCGACCGGUUGAUG 192 0.1634 −33.63 GGCGC UGAU ATCACATCAACCGGT 57 GCGCCUCCGGUUGAUGU 193 0.15877 −33.12 GGCGC GAU ATCACATCAACCGGT 58 GCGCCAGCGGUUGAUG 194 0.029249 −33.4 GGCGC UGAU ATCACATCAACCGGT 59 GCGCCACGGGUUGAUG 195 0.12208 −34.13 GGCGC UGAU ATCACATCAACCGGT 60 GCGCCACCCGUUGAUGU 196 0.051622 −31.57 GGCGC GAU ATCACATCAACCGGT 61 GCGCCACCGCUUGAUGU 197 0.004914 −31.74 GGCGC GAU ATCACATCAACCGGTG 62 GCGCCACCGGAUGAUGU 198 0.032227 −33.79 GCGC GAU GAGTTTCTCATCTGTG 63 GGGCCACAGAUGAGAA 199 0.015879 −33.54 CCCC ACUC CCAGCTTCTGCCGTTT 64 GUUCAAACGGCAGAAG 200 0.037469 −33.17 GTAC CUGG CCAGCTTCTGCCGTTT 65 GUACUAACGGCAGAAG 201 0.059921 −32.92 GTAC CUGG CCAGCTTCTGCCGTTT 66 GUACAAACGGGAGAAG 202 0.032605 −33.43 GTAC CUGG TTCCTCCTCCAGCTTC 67 GCCAGAAGCUGGAGGA 203 0.000481 −35.94 TGCC GGAA TTCCTCCTCCAGCTTC 68 GGCACAAGCUGGAGGA 204 0.041538 −37.4 TGCC GGAA TTCCTCCTCCAGCTTC 69 GGCAGAACCUGGAGGA 205 0.047874 −37.5 TGCC GGAA TTCCTCCTCCAGCTTC 70 GGCAGAAGCAGGAGGA 206 0.050381 −38.61 TGCC GGAA TTCCTCCTCCAGCTTC 71 GGCAGAAGCUCGAGGA 207 0.006459 −36.92 TGCC GGAA CCGGTTGATGTGATGG 72 GCACCCAUCACAUCAAC 208 0.03967 −33.31 GAGC CGG CCGGTTGATGTGATGG 73 GCUCCCUUCACAUCAAC 209 0.033426 −32.52 GAGC CGG CCGGTTGATGTGATGG 74 GCUCCCAACACAUCAAC 210 0.035651 −33.04 GAGC CGG CCGGTTGATGTGATGG 75 GCUCCCAUCAGAUCAAC 211 0.03209 −33.3 GAGC CGG GCAGCAAGCAGCACT 76 GGCAGUGUGCUGCUUG 212 0.004014 −32.46 CTGCC CUGC GCAGCAAGCAGCACT 77 GGCAGAGUGCAGCUUG 213 0.000219 −33.11 CTGCC CUGC GCTTGGGCCCACGCA 78 GCCCCAGCGUGGGCCCA 214 0.001487 −38.81 GGGGC AGC GCTTGGGCCCACGCA 79 GCCCCUGCCUGGGCCCA 215 0.003322 −36.77 GGGGC AGC GCTTCGTGGCAATGCG 80 GUGGCCCAUUGCCACGA 216 0.000463 −32.67 CCAC AGC GCTTGGGCCCACGCA 81 GCCCCUGCGUCGGCCCA 217 0 −37.12 GGGGC AGC AAGCTGGACTCTGGC 82 GAGUGGCCUGAGUCCA 218 0.010169 −33.02 CACTC GCUU TTCTTCTTCTGCTCGG 83 GAGACCGAGCAGAAGA 219 0.084395 −32.69 ACTC AGAA TTCTTCTTCTGCTCGG 84 GAGUCCGAGGAGAAGA 220 0.051852 −32.83 ACTC AGAA TTCTTCTTCTGCTCGG 85 GAGUCCGAGCUGAAGA 221 0.050685 −32.22 ACTC AGAA GAGTTTCTCATCTGTG 86 GGGGCACAGUUGAGAA 222 0.004503 −34.16 CCCC ACUC TTCCTCCTCCAGCTTC 87 GGCAGAAGGUGGAGGA 223 0.006035 −38.83 TGCC GGAA TTCCTCCTCCAGCTTC 88 GGCAGAAGCAGGAGGA 224 0.011364 −38.61 TGCC GGAA AGCAGAAGAAGAAGG 89 GGAGCCCUUGUUCUUCU 225 0.007206 −29.83 GCTCC GCU AAGCTGGACTCTGGC 90 GAGUGGCCUGAGUCCA 226 0 −33.02 CACTC GCUU CCCTAGTCATTGGAGG 91 GACACCUCCAAUGACUA 227 0.053611 −32.19 TGAC GGG CCCTAGTCATTGGAGG 92 GUGACCUCCAAUGACUA 228 0.05399 −31.73 TGAC GGG CCCTAGTCATTGGAGG 93 GUCUCCUCCAAUGACUA 229 0.070404 −31.72 TGAC GGG CCCTAGTCATTGGAGG 94 GUCAGCUCCAAUGACUA 230 0.067678 −32 TGAC GGG CCCTAGTCATTGGAGG 95 GUCACGUCCAAUGACUA 231 0.03597 −32.63 TGAC GGG CCCTAGTCATTGGAGG 96 GUCACCACCAAUGACUA 232 0.025207 −32.11 TGAC GGG CCCTAGTCATTGGAGG 97 GUCACCUGCAAUGACUA 233 0.056019 −31.63 TGAC GGG CCCTAGTCATTGGAGG 98 GUCACCUCGAAUGACUA 234 0.065347 −32.23 TGAC GGG CCCTAGTCATTGGAGG 99 GUCACCUCCUAUGACUA 235 0.063769 −31.62 TGAC GGG CCCTAGTCATTGGAGG 100 GUCACCUCCAUUGACUA 236 0.052644 −31.77 TGAC GGG ATGGGGAGGACATCG 101 GUCAUCGAUGUCCUCCC 237 0.020295 −31.26 ATGTC CAU ATGGGGAGGACATCG 102 GAGAUCGAUGUCCUCCC 238 0.012126 −31.7 ATGTC CAU ATGGGGAGGACATCG 103 GACUUCGAUGUCCUCCC 239 0.007202 −30.92 ATGTC CAU ATGGGGAGGACATCG 104 GACAACGAUGUCCUCCC 240 0.010912 −31.44 ATGTC CAU ATGGGGAGGACATCG 105 GACAUGGAUGUCCUCCC 241 0.009292 −31.83 ATGTC CAU ATGGGGAGGACATCG 106 GACAUCCAUGUCCUCCC 242 0.006125 −30.27 ATGTC CAU ATGGGGAGGACATCG 107 GACAUCGUUGUCCUCCC 243 0.007805 −31.06 ATGTC CAU ATGGGGAGGACATCG 108 GACAUCGAAGUCCUCCC 244 0.010174 −31.64 ATGTC CAU ATGGGGAGGACATCG 109 GACAUCGAUCUCCUCCC 245 0.003595 −30.32 ATGTC CAU ATGGGGAGGACATCG 110 GACAUCGAUGACCUCCC 246 0.000206 −31.59 ATGTC CAU ATCACATCAACCGGTG 111 GGGCCACCGGUUGAUG 247 0.18977 −35.23 GCGC UGAU ATCACATCAACCGGTG 112 GCCCCACCGGUUGAUGU 248 0.13525 −32.17 GCGC GAU ATCACATCAACCGGTG 113 GCGGCACCGGUUGAUG 249 0.14749 −33.43 GCGC UGAU ATCACATCAACCGGT 114 GCGCGACCGGUUGAUG 250 0.13952 −33.63 GGCGC UGAU ATCACATCAACCGGT 115 GCGCCUCCGGUUGAUGU 251 0.13949 −33.12 GGCGC GAU ATCACATCAACCGGT 116 GCGCCAGCGGUUGAUG 252 0.031221 −33.4 GGCGC UGAU ATCACATCAACCGGT 117 GCGCCACGGGUUGAUG 253 0.14776 −34.13 GGCGC UGAU ATCACATCAACCGGT 118 GCGCCACCCGUUGAUGU 254 0.050539 −31.57 GGCGC GAU ATCACATCAACCGGT 119 GCGCCACCGCUUGAUGU 255 0.003982 −31.74 GGCGC GAU ATCACATCAACCGGTG 120 GCGCCACCGGAUGAUGU 256 0.015494 −33.79 GCGC GAU GAGTTTCTCATCTGTG 121 GGGCCACAGAUGAGAA 257 0.025334 −33.54 CCCC ACUC CCAGCTTCTGCCGTTT 122 GUUCAAACGGCAGAAG 258 0.062094 −33.17 GTAC CUGG CCAGCTTCTGCCGTTT 123 GUACUAACGGCAGAAG 259 0.080429 −32.92 GTAC CUGG CCAGCTTCTGCCGTTT 124 GUACAAACGGGAGAAG 260 0.032505 −33.43 GTAC CUGG TTCCTCCTCCAGCTTC 125 GCCAGAAGCUGGAGGA 261 0.00117 −35.94 TGCC GGAA TTCCTCCTCCAGCTTC 126 GGCACAAGCUGGAGGA 262 0.034381 −37.4 TGCC GGAA TTCCTCCTCCAGCTTC 127 GGCAGAACCUGGAGGA 263 0.059128 −37.5 TGCC GGAA TTCCTCCTCCAGCTTC 128 GGCAGAAGCAGGAGGA 264 0.05162 −38.61 TGCC GGAA TTCCTCCTCCAGCTTC 129 GGCAGAAGCUCGAGGA 265 0.007682 −36.92 TGCC GGAA CCGGTTGATGTGATGG 130 GCACCCAUCACAUCAAC 266 0.093725 −33.31 GAGC CGG CCGGTTGATGTGATGG 131 GCUCCCUUCACAUCAAC 267 0.075435 −32.52 GAGC CGG CCGGTTGATGTGATGG 132 GCUCCCAACACAUCAAC 268 0.091723 −33.04 GAGC CGG CCGGTTGATGTGATGG 133 GCUCCCAUCAGAUCAAC 269 0.070319 −33.3 GAGC CGG GCAGCAAGCAGCACT 134 GGCAGUGUGCUGCUUG 270 0.006754 −32.46 CTGCC CUGC GCAGCAAGCAGCACT 135 GGCAGAGUGCAGCUUG 271 0.000545 −33.11 CTGCC CUGC GCTTGGGCCCACGCA 136 GCCCCAGCGUGGGCCCA 272 0.004676 −38.81 GGGGC AGC GCTTGGGCCCACGCA 137 GCCCCUGCCUGGGCCCA 273 0.001918 −36.77 GGGGC AGC GCTTCGTGGCAATGCG 138 GUGGCCCAUUGCCACGA 274 0.001045 −32.67 CCAC AGC GCTTGGGCCCACGCA 139 GCCCCUGCGUCGGCCCA 275 0 −37.12 GGGGC AGC AAGCTGGACTCTGGC 140 GAGUGGCCUGAGUCCA 276 0.008891 −33.02 CACTC GCUU TTCTTCTTCTGCTCGG 141 GAGACCGAGCAGAAGA 277 0.091861 −32.69 ACTC AGAA TTCTTCTTCTGCTCGG 142 GAGUCCGAGGAGAAGA 278 0.062783 −32.83 ACTC AGAA TTCTTCTTCTGCTCGG 143 GAGUCCGAGCUGAAGA 279 0.044444 −32.22 ACTC AGAA GAGTTTCTCATCTGTG 144 GGGGCACAGUUGAGAA 280 0.0053 −34.16 CCCC ACUC TTCCTCCTCCAGCTTC 145 GGCAGAAGGUGGAGGA 281 0.00714 −38.83 TGCC GGAA TTCCTCCTCCAGCTTC 146 GGCAGAAGCAGGAGGA 282 0.019945 −38.61 TGCC GGAA AGCAGAAGAAGAAGG 147 GGAGCCCUUGUUCUUCU 283 0.007996 −29.83 GCTCC GCU AAGCTGGACTCTGGC 148 GAGUGGCCUGAGUCCA 284 0.006102 −33.02 CACTC GCUU

KMC experiments were then performed to investigate the kinetics of strand invasion in the presence of PAM-distal mismatches. In all cases (1000 trials each), the guide RNAs remain quite stably bound even when there are mismatches (i.e., are not observed to completely melt off) and are often able to quickly bypass these sites to complete full invasion (FIG. 5C and FIGS. 14A-14C), although the mean first passage time of total strand invasion varied significantly depending on the position of the mismatch site (FIGS. 14A-14C). The R-loops are quite stable during invasion (FIG. 5A), as the sgRNAs are often able to remain fully invaded even in the presence of multiple mismatches. The results qualitatively resemble those of earlier in vitro studies of dCas9/Cas9 binding and cleavage on mismatched targets. However, in the case of tru-gRNAs (FIG. 5B), the R-loops are often trapped behind the mismatch sites. The mean first passage time across mismatches is similar for both sgRNAs and tru-gRNAs (FIGS. 14A-14C), but an inspection of the time courses for the KMC reveals that, because of the inherent volatility of the R-loop for tru-gRNAs, tru-gRNAs are often quickly ‘re-trapped’ behind the mismatch (FIG. 5C). For sgRNAs, this re-trapping is much less frequent. Hence, in combination with AFM imaging, the results of the KMC experiments suggest that the origin of increased tru-gRNA specificity lies not in discrimination during binding but rather in the volatility of its R-loop (FIG. 4D) such that it becomes repeatedly trapped behind mismatches even after initially bypassing them, making Cas9 less likely to assume the active conformation. For sgRNAs, once a mismatch is bypassed it can remain fully invaded with relatively little perturbation, suggesting a mechanism of mismatch tolerance.

Example 8 Stabilities of the Guide RNA Interaction with the 14th-17th Positions of the Protospacer are Correlated with Experimental Off-Target Cas9 Cleavage Rates, while Overall Guide RNA—Protospacer Binding Energies are not

To verify whether the stabilities of the R-loop at or near the 16^(th) position of the protospacer—which was implicated by AFM studies to be connected to the conformational change in Cas9—are associated with Cas9 activity in vivo, we performed a kinetic Monte Carlo (KMC) analysis of R-loop stability on the sequences used by Hsu et al. (2013) Nature Biotechnology, 31, 827-832. The data set of Hsu et al. (2013) Nature Biotechnology, 31, 827-832 consisted of measurements of the cleavage frequency at fifteen different protospacer targets containing various point mutations vs. the guide RNA that were performed to investigate cleavage specificity by Cas9. This data set contained 136 protospacer-guide RNA pairs that possessed a single, isolated mismatch of type rG·dG, rC·dC, rA·dA, and rU·dT in the PAM-distal region (Table 4), which we investigated using KMC methods initiated at R-loop size m=10 to simulate invasion. The inclusion of a single mismatched site from this set decreased the magnitude of their overall guide RNA—protospacer binding free energy on average by about only 6% relative to perfectly matched targets although, as mentioned, there was a wide distribution Cas9 cutting frequencies observed for these guide-RNA protospacer pairs whose origin was not obvious.

The mean fraction of time the RNA was bound stably to each site of the protospacer was determined for each guide RNA over 1000 trials, which was then correlated to the maximum-likelihood estimated cleavage activity of Cas9 (Table 4, FIG. 6 , and FIG. 15 ). A moderate (0.433) but statistically significant (p<1×10⁻⁶) correlation was found between guide RNA stability at the 16^(th) protospacer position and reported off-target cleavage activity. Notably, no statistically significant correlation was found between cleavage rate and the predicted DNA:RNA binding energies alone (0.0786; p=0.3631) (FIGS. 6A and 6B). In addition to R-loop stability at the 16^(th) position, a significant correlation is also found for stability the 17^(th) protospacer site and reported cleavage (Table 5), but this was not the case for sites ≥18^(th) site (FIG. 6 ). While the kinetic Monte Carlo model presented here is based on a relatively simple model of strand invasion, these results further suggest that stability of the 16^(th)-17^(th) sites of the protospacer, and hence the concomitant conformational changes we observed, are associated with Cas9 cleavage activity in vivo (FIG. 4D).

TABLE 5 Correlations between experimental (Hsu et al. (2013) Nature Biotechnology, 31, 827-832) cutting frequencies at target sites containing a single rG · dG, rC · dC, rA · dA, and rU · dT mismatch in the PAM-distal region (≥10^(th) protospacer site)^(a) and measures of guide RNA-protospacer stability log₁₀ Correlation (p-value) coefficient Hsu et al. (2013) estimated −0.4400 (0.0786) cutting frequency vs. guide RNA-protospacer binding energy^(b) Hsu et al. estimated cutting −5.8258 0.3990 frequency vs. position of mismatch site Hsu et al. m = 14 −9.5550 0.5078 estimated cutting m = 15 −7.4854 0.4522 frequency vs. m = 16 −6.9510 0.4333 fractional time m = 17 −3.9270 0.3191 guide RNA m = 18 −0.7639 (0.1159) bound at sites ≥ the m = 19 −0.5546 (0.1058) m^(th) protospacer site m = 20 −0.2346 (−0.0176) in a simulated R-loop (KMC)^(c) ^(a)n = 136. ^(b)See Table 4 for details. ^(c)See text for details. Max(t) = 100.

We limited most of our analysis to interactions with the 16^(th)-18^(th) nucleotides of the protospacer because of the observed structural differences between dCas9 with tru-gRNAs and sgRNA. However, we also observe an increase of the strength and statistical significance of the correlations between cleavage and the stability of the 14^(th) and 15^(th) protospacer sites (FIG. 6 ), with greatest significance for the correlation at the 14^(th) site. Because the R-loop is a dynamic structure (FIG. 4D), it is possible that interactions with these sites are those critical ones believed to be responsible for DNA cleavage. Truncation of the guide RNA by 4 or 5 nucleotides may abolish cleavage activity by sufficiently destabilizing the R-loop at the 14^(th) or 15^(th) position in much the same way that the tru-gRNA destabilized the R-loop at the 16^(th)-17^(th) sites. However, because in our model 14^(th) and 15^(th) sites are necessarily invaded whenever the 16^(th) site is bound by sgRNA, it is likely that these positions are additionally informative because they are also more strongly anti-correlated with the probability of sgRNA dissociation from the duplex prior to bypassing the mismatched site (FIG. 6Ai and FIG. 16B), another mechanism by which cleavage would fail to occur. At present, there is no crystallographic evidence which directly relates strand invasion to the observed conformational change believed to authorize cleavage. However, based on the evidence provided by AFM experiments presented here and the results of the kinetic Monte Carlo simulations, we conclude that stability of the guide gRNA at the 14^(th)-17^(th) sites of the protospacer during invasion is critical for this conformational change and, ultimately, the specificity of Cas9 cleavage.

Furthermore, the R-loop as a dynamic structure in competition between strand invasion and DNA re-annealing can be useful in understanding mechanisms of off-target cleavage and mismatch tolerance. No statistically significant correlation was found between cleavage rate and the predicted DNA-RNA binding energies alone (FIG. 6B), suggesting that the kinetics of strand invasion can be considered when attempting to determine Cas9 activity at off-target sites. While cleavage is abolished when 4 or 5 nucleotides are truncated from the guide RNA, Cas9 is still able to cleave DNA with up to 6 distal-mismatch sites. Transient, non-specific interactions at these PAM-distal sites could sufficiently stabilize the conformational shifts necessary for cleavage. Since we see minority populations of dCas9-sgRNA at partial protospacer sites with similar structures to those at the full protospacer (yellow, FIG. 3C(i)), this population may represent the fraction of Cas9 in a transiently-stabilized active conformation. As such, this population may be responsible for off-target cleavage.

While Cas9/dCas9 binding specificity is largely determined by interactions with the PAM-proximal region, DNA cleavage specificity is likely governed by a conformational change to an activated structure that is stabilized by guide RNA interactions at the 14^(th)-17^(th) bp region of the protospacer (FIG. 4D). Kinetic Monte Carlo experiments reveal that the R-loop formed during strand invasion of the guide RNA can be quite a dynamic structure even when the guide RNA remains stably bound, which suggests a mechanism for the improved specificity of tru-gRNAs, and an origin of off-target cleavage via transient stability of the guide RNA-protospacer at the critical region around mismatched sites. The proposed mechanisms for the effects of each of the sgRNA variants on Cas9/dCas9 specificity are summarized in FIG. 7 .

Using AFM, hp-gRNAs were found to significantly weakened or abolished specific binding at homeologous targets. hp-gRNAs may be valuable for modulating dCas9 binding affinity and specificity in their potential applications in biology and medicine. Specifically, based on the narrow geometry of the Cas9 binding channel, the presence of an unopened hairpin at mismatched protospacers may inhibit the conformational change by Cas9 to the active state. The opening of the hairpin in hp-gRNAs upon binding could also be used as a binding-dependent signal in vivo, for example, to nucleate dynamic DNA/RNA structures only upon binding to specific sites.

Earlier guide RNA truncation studies raised the question of why do natural Cas9 systems employ a crRNA which targets 20 bp protospacer sites when only a guide sequence of 16 nucleotides is required for cleavage and the additional nucleotides (>18) do not improve cleavage specificity in vivo. These results suggest that presence of the ‘extra’ 5′-nucleotides which bind to the 19^(th) and 20^(th) protospacer sites buffer this transient re-annealing at the critical 14^(th)-17^(th) sites of the protospacer, allowing efficient conformational change to the active state and subsequent cleavage to occur. The results of AFM and KMC experiments suggest that stability of the guide RNA at these sites shifts the equilibrium structure of Cas9 toward the active conformation upon full invasion (FIG. 4A), while the volatility of R-loops for ‘truncated’ guide RNAs reduces the pressure to shift the equilibrium to the active state. The promiscuous activity of Cas9 with sgRNAs vs. tru-gRNAs might also hold evolutionary advantages in its role as an agent of adaptive immunity in prokaryotes to invasive DNA, since the DNA of invading phages undergo rapid point mutations at sites targeted by Cas9 in order to avoid cleavage.

The design of guide RNA sequences for Cas9/dCas9 applications in vivo has focused primarily on avoiding targets with multiple sites with similar sequences in the genome. However, a recent study exploring off-target cleavage found that current methods for predicting off-target activity were largely ineffective. The stability of the R-loop during invasion correlates with off-target cleavage rates significantly better than guide RNA-protospacer binding energies alone or the position of the mismatch (another important criteria used in guide RNA design, Table 3). The stability of the R-loop at shorter times after the initiation of invasion was correlated with experimental cleavage rate much better than was the long-term stability in the KMC experiments (FIG. 16A), suggesting that the kinetics of strand invasion is a factor in off-target activity prediction.

Example 9 In Vivo Testing

Optimized gRNA activity was tested in living cells to investigate dCas9 binding specificity. Several hairpin gRNAs (hp-gRNAs) were designed for each of four target locations (protospacers) in the human genome (FIGS. 17 and 18 ). One was in the Dystrophin gene (FIGS. 19-23 ), another was in EMX1 gene (FIGS. 24-29 and 44 ), and two targets were in the VEGFA gene, labeled VEGFA1 (FIGS. 30-37 ) and VEGFA3 (FIGS. 38-43 ). All experiments were done in HEK293T cells.

Additional nucleotides (nt) were added to the 5′-end of full guide RNA (gRNAs, full length 20 nt) and designed to form Hairpins and secondary structures by hybridizing with the 5′-protospacer-targeting nucleotides, or nucleotides in the middle or the 3′-end of the protospacer-targeting region, in order to modulate binding and cleavage activity of Cas9 to protospacers.

One secondary structure of a VEGFA1-targeting hp-gRNA was computationally designed using the methods described herein to prevent binding at a known off-target site while allowing binding to the full protospacer (FIG. 44A-44C). The hp-gRNA was selected to have a binding lifetime greater than or equal to that of the full gRNA at the on-target site, and a binding lifetime less than or equal to that of the full-length gRNA at the top 3 off-target sites. Other 5′-structures were designed to include dG-rU wobble pairs to modulate the energetics of the secondary structures of the hp-gRNAs, or added to the end of truncated gRNAs (tru-gRNAs, <20 nt) which themselves have been shown to promote higher specificity of Cas9 activity.

Cell work. For the deep sequencing analysis, 293T cells were transfected with plasmids that expressed Cas9 and a gRNA of interest. The cells were incubated for 4 days, allowing for Cas9 and the gRNA to exert their maximum activity. The cells were then harvested and their genomic DNA was purified. gRNAs that were very well-characterized in the literature (i.e., their ontarget and off-target sites were known) were used.

Surveyor Assay. Compared to Deep-Sequencing, the surveyor assay is lower in throughput and less sensitive. However the surveyor assay is faster and less technical in data analysis, providing gel images. Thus surveyors were done as a first pass, and the best conditions were analyzed in triplicated with Deep-sequencing. Both DeepSequencing and Surveyor are methods to quantify mutational events caused by Cas9+gRNA.

The cell work for Surveyor was the same as described above. After genomic DNA was purified, primers were designed to amplify the targeted site. A pool of 200 k cells was used in this experiment and each one of them had a different mutation since DNA repair is stochastic. The site across 200 k cells was amplified to generate a heterogenous PCR product: some amplicons had deletions, some had insertions, and some were wild-type and unmodified, due to each cell stochastically (i.e. randomly, error prone) repairing Cas9 cut sites.

The heterogenous-PCR pool was heated and repaired, and in some cases different strands annealed to each other: a wild-type DNA strand might bind to DNA with an insertion, or an insertion might bind to a deletion. When this happens a little “bubble” formed and this structure is called a DNA heteroduplex (see FIG. 46 ).

The surveyor nuclease was used to detect these heteroduplexes by digesting and cleaving them. DNA cleavage was then a proxy for Cas9's mutational activity. The PCR pool was separated on a gel and the intensity of these digested bands was used to quantify the rate of Cas9 activity.

Deep Sequencing. Primers were designed to amplify these known targets/offtargets. A high-fidelity polymerase was used in this PCR. Illumina adapters were also present on these primers such that they could be barcoded and loaded onto the Illumina Mi-Seq platform. The # of hairpins, # of targets, # of offtargets, sequencing coverage, etc. are described in the figures and brief description of drawings. Good coverage was obtained across samples used in the analysis. The average number of reads/sample was 20,000. The sample with fewest # of reads was 1,700. A very small number of targets did not generate enough aligned reads and were not included in the analysis

The resulting sequencing data was analyzed using the CRISPResso software (Pinello et al. Nat Biotechnol. (2016) 34(7):695-697)), which aligns deep-sequencing reads with specific sites of known off-target or on-target locations. This software's results was compared with in-house scripts, in which global alignment of the Deep-sequencing reads with the human genome was performed, and correlated very well. Mutational rates were quantified using CRISPResso and the resulting data was displayed in the displayed histograms for each target gene.

Designs were first tested using Surveyor assays to test for indels after Cas9 and hp-gRNA expression in HEK cells at the target site and off-target sites known to be targeted using the standard gRNAs (see Table 6). Activity at these sites compared to the standard gRNA and truncated gRNAs (tru-gRNAs). These are shown below as gels showing cleavage by Surveyor nuclease of PCR'ed genomic DNA, where cleavage indicates mutagenesis by Cas9.

TABLE 6 Protospacers Genomic Targets Dystrophin 1 on-target, 1 off-on-target EMX1 1 on-target, 7 off-target VEGFA1 1 on-target, 10 off-target VEGFA3 1 on-target, 22 off-target

The most promising hp-gRNA designs were chosen for additional quantitative analysis using next-gen sequencing to evaluate Cas9 activity at on- and off-target sites in HEK cells. Specificity was defined as on-target hits/sum (off-target hits).

While Cas9 activity was generally equal to or slightly decreased when using hp-gRNAs, each hp-gRNAs selected for Deep-Seq experiments showed enhanced specificity over full gRNAs, and in most cases were equal to or greater than tru-gRNAs in terms of specificity.

In one case, a hp-gRNA hairpin targeting EMX1 exhibited >6000-fold improvement in specificity over full gRNA (vs. tru-gRNA with ˜ 100-fold improvement over gRNA). The VEGFA1-targeting hp-gRNA with a computationally-designed secondary structure using an in-house algorithm greatly outperformed the tru-gRNA activity in terms of specificity (18-fold vs. 3-fold improvement over gRNA). These hp-gRNAs were tested in conjunction with S. pyogenes Cas9. FIG. 44A-44C shows Surveyor assays of EMX1-targeting hp-gRNAs with Cas9 from S. aureous exhibiting on-target activity and no detectable off-target activity, in contrast to tru-gRNAs which show significant off-target activity.

Example 10 hp-gRNA for CRISPR/Cpf1 System

Experiments were designed to reproduce the results of Kleinstiver et al., Nat. Biotech. (2016) 34:869-874. Kleinstiver et al. used full-length gRNAs to show that Lachnospiraceae Cpf1 is susceptible to cut at off-target sites with mismatches at the 8-9 nucleotides in addition to PAM-distal sites, by using gRNAs which had mismatches with the target site at different locations (FIG. 47 ). In this example, hairpin guide RNAs used with the Type V CRISPR-Cas system CRISPR-Cpf1 were designed and tested as described above using the methods of the present invention.

To test off-target activity of Cpf1 with and without the additional secondary structure elements, the DNMT1 gene (TTTC CTGATGGGTCCATGTCTGTTACTC (SEQ ID NO: 330)) was targeted for cleavage by Cpf1. “Off-target activity” was tested by using guide RNAs which had a mismatched nucleotide at position 9, e.g. CTGATGGTgCATGTCTGTTA (SEQ ID NO: 331), using full-length guide RNAs 20 nucleotides long or truncated gRNAs 17 nucleotides long CTGATGGTgCATGTCTG (SEQ ID NO: 332). 9 nucleotide long secondary structure elements were added to the 3′-end of the Cpf1 guide RNAs to hybridize with the segment of the guide RNA surrounding the mismatched nucleotide, where in this case the ‘linker’ element were comprised of the 4 3′-nt of the protospacer-targeting segment, i.e., CTGATGGTgCATGTCT GTTA AGACATGcACCA (SEQ ID NO: 333) and CTGATGGTgCATG TCTG CATGcACCA (SEQ ID NO: 334). A Surveyor assay shows that that inclusion of these additional 3′-elements decreased or abolished the off-target activity at the DNMT1 site exhibited by the full or truncated gRNAs.

hp-gRNAs were designed with an “internal” hairpin design in which the PAM-distal 4 nucleotide served as the loop. The hairpin was added to the 3′-end of the gRNA. Table 7 shows the sequences of the hp-gRNA with a space in the sequences that separates this region. The mismatch is shown in lower case.

Surveyor results of these hp-gRNAs are shown in FIG. 48 and show that the addition of the hairpin to the 3′-end abolished off-target activity. Lane 1 shows the control; lane 2 shows a full-length gRNA containing a mismatched nucleotide at position 9; lane 3 shows the full-length gRNA containing a mismatched nucleotide at position 9 and an additional 3′-hairpin structure; lane 4 shows a truncated gRNA containing a mismatched nucleotide at position 9; and lane 5 shows the truncated gRNA containing a mismatched nucleotide at position 9 and an additional 3′-hairpin structure. The Surveyor primers used are also shown in Table 7.

Cpf1 tolerates mismatches at nucleotides 8-10 when using normal guide RNAs and cleaves DNA at those off-target sites (FIG. 47 ). As shown in FIG. 48 , the Cpf1 hp-gRNA were able to abolish the off-target activity shown in the Kleinstiver, while the truncated gRNAs could not.

TABLE 7 Surveyor Expected primers product Label Sequence size CN391 DNMT1 CTGGGACTCA 606 bp (forward) GGCGGGTCAC (SEQ ID  NO: 324) CN406 DNMT1 CCTCACACAA reverse CAGCTTCATG fixed TCAGC (SEQ ID NO: 325) Protospacer Sequences Label Sequence LbCpf1_ CTGATGGT 9mm_20nt_S gCATGTCT GTTA (SEQ ID NO: 326) LbCpf1_ CTGATGGTg 9mm_17nt_S CATG TCTG (SEQ ID NO: 327) LbCpf1_9mm_ CTGATGGTgCATGTCT G 20nt_hp_S TTA AGACATGcACCA (SEQ ID NO: 328) LbCpf1_9mm_ CTGATGGTgCATG TCTG 17nthpS CATGcACCA (SEQ ID NO: 329)

It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the invention, which is defined solely by the appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the invention, may be made without departing from the spirit and scope thereof.

For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

Clause 1. A method of generating an optimized guide RNA (gRNA), the method comprising: a) identifying a target region of interest, the target region of interest comprising a protospacer sequence; b) determining a polynucleotide sequence of a full-length gRNA that targets the target region of interest, the full-length gRNA comprising a protospacer-targeting sequence or segment; c) determining at least one or more off-target sites for the full-length gRNA; d) generating a polynucleotide sequence of a first gRNA, the first gRNA comprising the polynucleotide sequence of the full-length gRNA and a RNA segment, the RNA segment comprising a polynucleotide sequence having a length of M nucleotides that is complementary to a nucleotide segment of the protospacer-targeting sequence or segment, the RNA segment is at the 5′ end of the polynucleotide sequence of the full-length gRNA, the first gRNA optionally comprising a linker between the 5′ end of the polynucleotide sequence of the full-length gRNA and the RNA segment, the linker comprising a polynucleotide sequence having a length of N nucleotides, the first gRNA capable of invading the protospacer sequence and binding to a DNA sequence that is complementary to the protospacer sequence and forming a protospacer-duplex, and the first gRNA capable of invading an off-target site and binding to a DNA sequence that is complementary to the off-target site and forming an off-target duplex; e) calculating an estimate or computationally simulating the invasion kinetics and lifetime that the first gRNA remains invaded in the protospacer and off-target site duplexes, wherein the dynamics of invasion are estimated nucleotide-by-nucleotide by determining the energetic differences between further invasion of a different gRNA and re-annealing of the first gRNA to the DNA sequence that is complementary to the protospacer sequence; f) comparing the estimated lifetimes at the protospacer and/or off-target sites of the first gRNA with the estimated lifetimes of the full-length gRNA or a truncated gRNA (tru-gRNA) at the protospacer and/or off-target sites; g) randomizing 0 to N nucleotides in the linker and 0 to M nucleotides in the first gRNA and generating a second gRNA and repeating step (e) with the second gRNA; h) identifying an optimized gRNA based on a gRNA sequence that satisfy a design criteria; and i) testing the optimized gRNA in vivo to determine the specificity of binding.

Clause 2. A method of generating an optimized guide RNA (gRNA), the method comprising: a) identifying a target region of interest, the target region of interest comprising a protospacer sequence; b) determining a polynucleotide sequence of a full-length gRNA that targets the target region of interest, the full-length gRNA comprising a protospacer-targeting sequence or segment; c) determining at least one or more off-target sites for the full-length gRNA; d) generating a polynucleotide sequence of a first gRNA, the first gRNA comprising the polynucleotide sequence of the full-length gRNA and a RNA segment, the RNA segment comprising a polynucleotide sequence having a length of M nucleotides that is complementary to a nucleotide segment of the protospacer-targeting sequence or segment, the RNA segment is at the 3′ end of the polynucleotide sequence of the full-length gRNA, the first gRNA optionally comprising a linker between the 3′ end of the polynucleotide sequence of the full-length gRNA and the RNA segment, the linker comprising a polynucleotide sequence having a length of N nucleotides, the first gRNA capable of invading the protospacer sequence and binding to a DNA sequence that is complementary to the protospacer sequence and forming a protospacer-duplex, and the first gRNA capable of invading an off-target site and binding to a DNA sequence that is complementary to the off-target site and forming an off-target duplex; e) calculating an estimate or computationally simulating the invasion kinetics and lifetime that the first gRNA remains invaded in the protospacer and off-target site duplexes, wherein the dynamics of invasion are estimated nucleotide-by-nucleotide by determining the energetic differences between further invasion of a different gRNA and re-annealing of the first gRNA to the DNA sequence that is complementary to the protospacer sequence; f) comparing the estimated lifetimes at the protospacer and/or off-target sites of the first gRNA with the estimated lifetimes of the full-length gRNA or a truncated gRNA (tru-gRNA) at the protospacer and/or off-target sites; g) randomizing 0 to N nucleotides in the linker and 0 to M nucleotides in the first gRNA and generating a second gRNA and repeating step (e) with the second gRNA; h) identifying an optimized gRNA based on a gRNA sequence that satisfy a design criteria; and i) testing the optimized gRNA in vivo to determine the specificity of binding.

Clause 3. The method of clause 1 or 2, wherein the energetics of further invasion of a different gRNA is determined by determining the energetics of at least one of (I) breaking a DNA-DNA base-pairing, (II) forming an RNA-DNA base-pair, (III) energetic difference resulting from disrupting or forming different secondary structure within the uninvaded guide RNA, and (IV) forming or disrupting interactions between the displaced DNA strand that is complementary to the protospacer and any unpaired guide RNA nucleotides which are not involved in secondary structures.

Clause 4. The method of any one of clauses 1-3, wherein the energetics of re-annealing of the first gRNA to the DNA sequence that is complementary to the protospacer sequence is determined by determining the energetics of at least one of (I) forming a DNA-DNA base-pairing, (II) breaking an RNA-DNA base-pair, (III) energetic difference resulting from disrupting or forming different secondary structure within the newly uninvaded guide RNA, and (IV) forming or disrupting interactions between the displaced DNA strand that is complementary to the protospacer and any unpaired guide RNA nucleotides which are not involved in secondary structures.

Clause 5. The method of clause 3 or 4, further comprising determining the energetic considerations from at least one of (V) base-pairing across mismatches, (VI) interactions with the Cas9 protein, and/or (VII) additional heuristics, wherein the additional heuristics relate to binding lifetime, extent of invasion, stability of invading guide RNA, or other calculated/simulated properties of gRNA invasion to Cas9 cleavage activity.

Clause 6. The method of any one of clauses 1-5, wherein the full-length gRNA comprises between about 15 and 20 nucleotides.

Clause 7. The method of any one of clauses 1-5, wherein M is between 1 and 20.

Clause 8. The method of clause 7, wherein M is between 4 and 10.

Clause 9. The method of any one of clauses 1-8, wherein the RNA segment comprises between 2 and 15 nucleotides that complement the protospacer-targeting sequence.

Clause 10. The method of any one of clauses 1-9, wherein N is between 1 and 20.

Clause 11. The method of clause 10, wherein N is between 3 and 10.

Clause 12. The method of any one of clauses 1-11, wherein the RNA segment and/or protospacer-targeting sequence provide a secondary structure.

Clause 13. The method of clause 12, wherein the secondary structure is formed by partially hybridizing the protospacer-targeting sequence with the RNA segment.

Clause 14. The method of clause 13, wherein the secondary structure modulates DNA binding or cleavage by Cas9 by disrupting invasion of the protospacer duplex or off-target duplex by the optimized gRNA.

Clause 15. The method of any one of clauses 12-14, wherein the secondary structure is formed by hybridizing all or part of the RNA segment to nucleotides in the 5′-end of the protospacer-targeting sequence or segment, nucleotides in the middle of the protospacer-targeting sequence or segment, and/or nucleotides in the 3′-end of the protospacer-targeting sequence or segment.

Clause 16. The method of any one of clauses 12-15, wherein the secondary structure is a hairpin.

Clause 17. The method of any one of clauses 12-16, wherein the secondary structure is stable at room temperature or 37° C.

Clause 18. The method of any one of clauses 12-17, wherein the overall equilibrium free energy of the secondary structure is less than about 2 kcal/mol at room temperature or 37° C.

Clause 19. The method of any one of clauses 1-18, wherein the RNA segment hybridizes or forms non-canonical base pairs with at least two nucleotides of the protospacer-targeting sequence or segment.

Clause 20. The method of clause 19, wherein the non-canonical base pair is rU-rG.

Clause 21. The method of any one of clauses 1-20, wherein the optimized gRNA is used with a CRISPR/Cas9-based system or CRISPR/Cpf1-based system in a cell.

Clause 22. The method of any one of clauses 1-21, wherein the secondary structure protects the optimized gRNA within the CRISPR/Cas9-based system or CRISPR/Cpf1-based system to prevent degradation within the cell.

Clause 23. The method of any one of clauses 1-22, wherein 1-20 nucleotides are randomized in the linker.

Clause 24. The method of any one of clauses 1-23, wherein 1-20 nucleotides are randomized in the RNA segment.

Clause 25. The method of any one of clauses 1-24, wherein step (g) is repeated X number of times, thereby generating X number of gRNAs and repeating step (e) with each X number of gRNAs, wherein X is between 0 to 20.

Clause 26. The method of any one of clauses 1-25, wherein the invasion kinetics and lifetime are calculated using kinetic Monte Carlo method or Gillespie algorithm.

Clause 27. The method of any one of clauses 1-26, wherein the invasion kinetics is the rate at which the guide RNA invades the protospacer duplex to full invasion such that the protospacer is completely invaded and/or the rate at which the segment of protospacer DNA bound to the gRNA expands as it is displaced from its complementary strand and bound to the gRNA nucleotide-by-nucleotide from its PAM proximal region through to full invasion.

Clause 28. The method of any one of clauses 1-27, wherein the design criteria comprises specificity, modulation of binding lifetime, and/or estimated cleavage specificity.

Clause 29. The method of clause 28, wherein the design criteria comprises an optimized gRNA having a binding lifetime greater than or equal to the binding lifetime of a full-length gRNA to the on-target site and/or a binding lifetime less than or equal to the binding lifetime of a full-length gRNA to an off-target site.

Clause 30. The method of clause 29, wherein the design criteria comprises an optimized gRNA having a binding lifetime less than or equal to the binding lifetime of a full-length gRNA to at least three off-target sites, wherein the off-target sites are predicted to be the closest off-target sites or predicted to have the highest identity to the on-target sites.

Clause 31. The method of clause 28, wherein the design criteria comprises a lifetime or cleavage rate at an off-target site that is less than or equal to the lifetime or cleavage rate of a full-length gRNA or truncated gRNA at the off-target site and/or a predicted on-target activity rate that is greater than 10% of the predicted on-target activity rate of a full-length gRNA or truncated gRNA.

Clause 32. The method of any one of clauses 1-31, wherein the optimized gRNA is tested in step i) using surveyor assay, next-gen sequencing techniques, or GUIDE-Seq.

Clause 33. The method of any one of clauses 1-32, wherein the optimized gRNA is designed to minimize binding at an off-target site and allow binding to a protospacer sequence.

Clause 34. The method of any one of clauses 1-33, wherein the off-target site is a known or predicted off-target site.

Clause 35. The method of any one of clauses 1-34, wherein the full-length gRNA targets a mammalian gene.

Clause 36. The method of any one of clauses 1-35, wherein the target gene comprises an endogenous target gene or a transgene.

Clause 37. The method of any one of clauses 1-36, wherein the target gene comprises a disease-relevant gene.

Clause 38. The method of any one of clauses 1-37, wherein the target gene is a DMD, EMX1, or VEGFA gene.

Clause 39. The method of clause 38, wherein the VEGFA gene is VEGFA1 or VEGFA3.

Clause 40. An optimized gRNA generated by the method of any one of clauses 1-39.

Clause 41. The optimized gRNA of clause 40, wherein the gRNA can discriminate between on- and off-target sites with minimal thermodynamic energetic differences between the sites.

Clause 42. The optimized gRNA of clause 40 or 41, wherein the optimized gRNA modulates strand invasion into the protospacer.

Clause 43. The optimized gRNA of any one of clauses 40-42, wherein the optimized gRNA comprises a nucleotide sequence of at least one of SEQ ID NOs: 149-315, 321-323, and 326-329.

Clause 44. An isolated polynucleotide encoding the optimized gRNA of any one of clauses 40-43.

Clause 45. A vector comprising the isolated polynucleotide of clause 44.

Clause 46. A cell comprising the isolated polynucleotide of clause 44 or the vector of clause 45.

Clause 47. A kit comprising the isolated polynucleotide of clause 44, the vector of clause 45, or the cell of clause 46.

Clause 48. A method of epigenomic editing in a target cell or a subject, the method comprising contacting a cell or a subject with an effective amount of the optimized gRNA molecule of any one of clauses 40-43 or the isolated polynucleotide of clause 44 and a fusion protein, the fusion protein comprising a first polypeptide domain comprising a nuclease-deficient Cas9 and a second polypeptide domain having an activity selected from the group consisting of transcription activation activity, transcription repression activity, nuclease activity, transcription release factor activity, histone modification activity, nucleic acid association activity, DNA methylase activity, and direct or indirect DNA demethylase activity.

Clause 49. A method of site specific DNA cleavage in a target cell or a subject, the method comprising contacting a cell or a subject with an effective amount of the optimized gRNA molecule of any one of clauses 40-43 or the isolated polynucleotide of clause 44 and a fusion protein or Cas9 protein, the fusion protein comprising a first polypeptide domain comprising a nuclease-deficient Cas9 and a second polypeptide domain having an activity selected from the group consisting of transcription activation activity, transcription repression activity, nuclease activity, transcription release factor activity, histone modification activity, nucleic acid association activity, DNA methylase activity, and direct or indirect DNA demethylase activity.

Clause 50. A method of genome editing in a cell, the method comprising administering to the cell an effective amount of the optimized gRNA molecule of any one of clauses 40-43 or the isolated polynucleotide of clause 44 and a fusion protein, the fusion protein comprising a first polypeptide domain comprising a nuclease-deficient Cas9 and a second polypeptide domain having an activity selected from the group consisting of transcription activation activity, transcription repression activity, nuclease activity, transcription release factor activity, histone modification activity, nucleic acid association activity, DNA methylase activity, and direct or indirect DNA demethylase activity.

Clause 51. The method of clause 50, wherein the genome editing comprises correcting a mutant gene or inserting a transgene.

Clause 52. The method of clause 51, wherein correcting a mutant gene comprises deleting, rearranging, or replacing the mutant gene.

Clause 53. The method of any one of clauses 51 or 52, wherein correcting the mutant gene comprises nuclease-mediated non-homologous end joining or homology-directed repair.

Clause 54. A method of modulating gene expression in a cell, the method comprising contacting the cell with an effective amount of the optimized gRNA molecule of any one of clauses 40-43 or the isolated polynucleotide of clause 44 and a fusion protein, the fusion protein comprising a first polypeptide domain comprising a nuclease-deficient Cas9 and a second polypeptide domain having an activity selected from the group consisting of transcription activation activity, transcription repression activity, nuclease activity, transcription release factor activity, histone modification activity, nucleic acid association activity, DNA methylase activity, and direct or indirect DNA demethylase activity.

Clause 55. The method of clause 54, wherein the gene expression of the at least one target gene is modulated when gene expression levels of the at least one target gene are increased or decreased compared to normal gene expression levels for the at least one target gene.

Clause 56. The method of clause 54 or 55, wherein the fusion protein comprises a dCas9 domain and a transcriptional activator.

Clause 57. The method of clause 56, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 2.

Clause 58. The method of clause 54 or 55, wherein the fusion protein comprises a dCas9 domain and a transcriptional repressor.

Clause 59. The method of clause 58, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:3.

Clause 60. The method of clause 54 or 55, wherein the fusion protein comprises a dCas9 domain and a site-specific nuclease.

Clause 61. The method of any one of clauses 48-60 wherein the optimized gRNA is encoded by a polynucleotide sequence and packaged into a lentiviral vector.

Clause 62. The method of clause 61, wherein the lentiviral vector comprises an expression cassette comprising a promoter operably linked to the polynucleotide sequence encoding the gRNA.

Clause 63. The method of clause 62, wherein the promoter operably linked to the polynucleotide encoding the optimized gRNA is inducible.

Clause 64. The method of any one of clauses 61-63, herein the lentiviral vector further comprises a polynucleotide sequence encoding the Cas9 protein or fusion protein.

Clause 65. The method of any one of clauses 48-64, wherein the at least one target gene is a disease-relevant gene.

Clause 66. The method of any one of clauses 48-65, wherein the target cell is a eukaryotic cell.

Clause 67. The method of any one of clauses 48-66, wherein the target cell is a mammalian cell.

Clause 68. The method of any one of clauses 48-67, wherein the target cell is a HEK293T cell.

Appendix-Sequences Streptococcus pyogenes Cas 9 (with D10A, H840A) (SEQ ID NO: 1) MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDR HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDA IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSV KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLAS HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD dCas9p300 Core: (Addgene Plasmid 61357) amino acid sequence; 3X “Flag” Epitope, Nuclear Localization Sequence, Streptococcus pyogenes Cas9 (D10A, H840A), p300 Core Effector, “HA” Epitope (SEQ ID NO: 2) MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGRGMDKKY SIGL A IGTNSVGWA VITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFH RLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLR KKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKS RRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLA EDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDA ILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALV RQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPI LEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGEL HAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFD KNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAF LSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVE ISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDI VLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGW GRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHD DSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQ TVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRE RMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNG RDMYVDQELDINRLSDYDVD A IVPQSFLKDDSIDNKVLTR SDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRM NTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREIN NYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVR KMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRK RPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTE VQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGE LQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFV EQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRD KPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTST KEVLDATLIHQSITGLYETRIDLSQLGGDPIAGSKASPKK KRKVGRAIFKPEELRQALMPTLEALYRQDPESLPFRQPVD PQLLGIPDYFDIVKSPMDLSTIKRKLDTGQYQEPWQYVDD IWLMFNNAWLYNRKTSRVYKYCSKLSEVFEQEIDPVMQSL GYCCGRKLEFSPQTLCCYGKQLCTIPRDATYYSYQNRYHF CEKCFNEIQGESVSLGDDPSQPQTTINKEQFSKRKNDTLD PELFVECTECGRKMHQICVLHHEIIWPAGFVCDGCLKKSA RTRKENKFSAKRLPSTRLGTFLENRVNDFLRRQNHPESGE VTVRVVHASDKTVEVKPGMKARFVDSGEMAESFPYRTKAL FAFEEIDGVDLCFFGMHVQEYGSDCPPPNQRRVYISYLDS VHFFRPKCLRTAVYHEILIGYLEYVKKLGYTTGHIWACPP SEGDDYIFHCHPPDQKIPKPKRLQEWYKKMLDKAVSERIV HDYKDIFKQATEDRLTSAKELPYFEGDFWPNVLEESIKEL EQEEEERKREENTSNESTDVTKGDSKNAKKKNNKKTSKNK SSLSRGNKKKPGMPNVSNDLSQKLYATMEKHKEVFFVIRL IAGPAANSLPPIVDPDPLIPCDLMDGRDAFLTLARDKHLE FSSLRRAOWSTMCMLVELHTQSQD Y PYDVPDYAS dCas9^(KRAB) (SEQ ID NO: 3) MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGRGMDKKY SIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEI FSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDE VAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFR GHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASG VDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQ YADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRY DEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDG GASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTF DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILT FRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKG ASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELT KVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLK EDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKD FLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDD KVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKS DGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIA NLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQ LQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQS FLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQ LLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETR QITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSD FRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLE SEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNF FKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRK VLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKD WDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLG ITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKL KGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFK YFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQL GGDSRADPKKKRKVASDAKSLTAWSRTLVTFKDVFVDFTR EEWKLLDTAQQILYRNVMLENYKNLVSLGYQLTKPDVILR LEKGEEPWLVEREIHQETHPDSETAFEIKSSVPKKKRKVA S Nm-dCas9300 Core: (Addgene Plasmid 61365) amino acid sequence; Neisseria meningitidis Cas9 (D16A, D587A, H588A, N611A), Nuclear Localization Sequence, p300 Core Effector, “HA” Epitope (SEQ ID NO: 5) MAAFKPNPINYILGL A IGIASVGWAMVEIDEDENPICLID LGVRVFERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLL RARRLLKREGVLQAADFDENGLIKSLPNTPWQLRAAALDR KLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLK GVADNAHALQTGDFRTPAELALNKFEKESGHIRNQRGDYS HTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLLM TQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWL TKLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQA RKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRAL EKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLK DRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKR YDEACAEIYGDHYGKKNTEEKIYLPPIPADEIRNPVVLRA LSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRKEIE KRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYE QQHGKCLYSGKEINLGRLNEKGYVEI AA ALPFSRTWDDSF NNKVLVLGSE A QNKGNQTPYEYFNGKDNSREWQEFKARVE TSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQF VADRMRLTGKGKKRVFASNGQITNLLRGFWGLRKVRAEND RHHALDAVVVACSTVAMQQKITRFVRYKEMNAFDGKTIDK ETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEA DTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSG QGHMETVKSAKRLDEGVSVLRVPLTQLKLKDLEKMVNRER EPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQV KAVRVEQVQKTGVWVRNHNGIADNATMVRVDVFEKGDKYY LVPIYSWQVAKGILPDRAVVQGKDEEDWQLIDDSFNFKFS LHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHK IGKNGILEGIGVKTALSFQKYQIDELGKEIRPCRLKKRPP VRSRADPKKKRKVEASGRAIFKPEELRQALMPTLEALYRQ DPESLPFRQPVDPQLLGIPDYFDIVKSPMDLSTIKRKLDT GQYQEPWQYVDDIWLMFNNAWLYNRKTSRVYKYCSKLSEV FEQEIDPVMQSLGYCCGRKLEFSPQTLCCYGKQLCTIPRD ATYYSYQNRYHFCEKCFNEIQGESVSLGDDPSQPQTTINK EQFSKRKNDTLDPELFVECTECGRKMHQICVLHHEIIWPA GFVCDGCLKKSARTRKENKFSAKRLPSTRLGTFLENRVND FLRRQNHPESGEVTVRVVHASDKTVEVKPGMKARFVDSGE MAESFPYRTKALFAFEEIDGVDLCFFGMHVQEYGSDCPPP NQRRVYISYLDSVHFFRPKCLRTAVYHEILIGYLEYVKKL GYTTGHIWACPPSEGDDYIFHCHPPDQKIPKPKRLQEWYK KMLDKAVSERIVHDYKDIFKQATEDRLTSAKELPYFEGDF WPNVLEESIKELEQEEEERKREENTSNESTDVTKGDSKNA KKKNNKKTSKNKSSLSRGNKKKPGMPNVSNDLSQKLYATM EKHKEVFFVIRLIAGPAANSLPPIVDPDPLIPCDLMDGRD AFLTLARDKHLEFSSLRRAOWSTMCMLVELHTQSQD YPYD VPDYAS 

1. A gRNA comprising a nucleotide sequence selected from SEQ ID NOs: 149-284 or encoded by a polynucleotide comprising a nucleotide sequence selected from SEQ ID NOs: 285-315, 321-323, and 326-329.
 2. A CRISPR/Cas-based base editing system comprising: the gRNA of claim 1; and a Cas protein or a fusion protein, the fusion protein comprising a first polypeptide domain comprising a nuclease-deficient Cas9 and a second polypeptide domain having an activity selected from the group consisting of transcription activation activity, transcription repression activity, nuclease activity, transcription release factor activity, histone modification activity, nucleic acid association activity, DNA methylase activity, and direct or indirect DNA demethylase activity.
 3. The CRISPR/Cas-based base editing system of claim 2, wherein the fusion protein comprises a dCas9 domain and a transcriptional activator, or wherein the fusion protein comprises a dCas9 domain and a transcriptional repressor, or wherein the fusion protein comprises a dCas9 domain and a site-specific nuclease.
 4. The CRISPR/Cas-based base editing system of claim 2, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 2 or
 3. 5. An isolated polynucleotide encoding the gRNA of claim
 1. 6. A vector comprising the isolated polynucleotide of claim
 5. 7. A cell comprising the isolated polynucleotide of claim
 5. 8. A kit comprising the isolated polynucleotide of claim
 5. 9. A method of site specific DNA cleavage in a target cell or a subject, the method comprising contacting the cell or the subject with an effective amount of the gRNA of claim 1 and a fusion protein or Cas9 protein, the fusion protein comprising a first polypeptide domain comprising a nuclease-deficient Cas9 and a second polypeptide domain having an activity selected from the group consisting of transcription activation activity, transcription repression activity, nuclease activity, transcription release factor activity, histone modification activity, nucleic acid association activity, DNA methylase activity, and direct or indirect DNA demethylase activity.
 10. A method of modulating gene expression in a cell, the method comprising contacting the cell with an effective amount of the gRNA of claim 1 and a fusion protein, the fusion protein comprising a first polypeptide domain comprising a nuclease-deficient Cas9 and a second polypeptide domain having an activity selected from the group consisting of transcription activation activity, transcription repression activity, nuclease activity, transcription release factor activity, histone modification activity, nucleic acid association activity, DNA methylase activity, and direct or indirect DNA demethylase activity.
 11. The method of claim 10, wherein the gene expression of at least one target gene is modulated when gene expression levels of the at least one target gene are increased or decreased compared to normal gene expression levels for the at least one target gene.
 12. The method of claim 10, wherein the fusion protein comprises a dCas9 domain and a transcriptional activator, or a dCas9 domain and a transcriptional repressor, or a dCas9 domain and a site-specific nuclease.
 13. The method of claim 12, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:3.
 14. The method of claim 10, wherein the gRNA is encoded by a polynucleotide sequence and packaged into a lentiviral vector.
 15. The method of claim 14, wherein the lentiviral vector comprises an expression cassette comprising a promoter operably linked to the polynucleotide sequence encoding the gRNA.
 16. The method of claim 15, wherein the lentiviral vector further comprises a polynucleotide sequence encoding the fusion protein.
 17. A method of epigenomic editing in a target cell or a subject, the method comprising contacting a cell or a subject with an effective amount of the gRNA of claim 1 and a fusion protein, the fusion protein comprising a first polypeptide domain comprising a nuclease-deficient Cas9 and a second polypeptide domain having an activity selected from the group consisting of transcription activation activity, transcription repression activity, nuclease activity, transcription release factor activity, histone modification activity, nucleic acid association activity, DNA methylase activity, and direct or indirect DNA demethylase activity.
 18. A method of genome editing in a cell, the method comprising administering to the cell an effective amount of the gRNA of claim 1 and a fusion protein, the fusion protein comprising a first polypeptide domain comprising a nuclease-deficient Cas9 and a second polypeptide domain having an activity selected from the group consisting of transcription activation activity, transcription repression activity, nuclease activity, transcription release factor activity, histone modification activity, nucleic acid association activity, DNA methylase activity, and direct or indirect DNA demethylase activity.
 19. The method of claim 19, wherein the genome editing comprises deleting a mutant gene, rearranging a mutant gene, replacing a mutant gene, or inserting a transgene.
 20. A method of treating a subject, the method comprising: administering to the subject a DNA targeting system comprising the gRNA of claim 1 and a fusion protein or Cas9 protein, the fusion protein comprising a first polypeptide domain comprising a nuclease-deficient Cas9 and a second polypeptide domain having an activity selected from the group consisting of transcription activation activity, transcription repression activity, nuclease activity, transcription release factor activity, histone modification activity, nucleic acid association activity, DNA methylase activity, and direct or indirect DNA demethylase activity. 